CA3005883A1 - Ratiometric biosensors and non-geometrically modulated fret - Google Patents

Abstract

The present subject matter provides ratiometric biosensors as well as compositions, devices, and methods comprising such biosensors.

Description

2 RATIOMETRIC BIOSENSORS AND
NON-GEOMETRICALLY MODULATED FRET
RELATED APPLICATIONS
This application claims benefit of priority to U.S. Provisional Application No.
62/257,850, filed November 20, 2015, U.S. Provisional Application No.
62/257,859, filed November 20, 2015, U.S. Provisional Application No. 62/257,863, filed November 20, 2015, and U.S. Provisional Application No. 62/257,796, filed November 20, 2015, the entire contents of each which are incorporated herein by reference.
INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING
The contents of the text file named "35327-521001WO_Sequence_Listing.txt", which was created on November 19, 2016 and is 390 KB in size, is hereby incorporated by reference in its entirety.
FIELD OF THE INVENTION
The present invention relates to compositions and methods for detecting compounds and determining the concentration thereof.
BACKGROUND
Determination of analyte concentrations using fluorescent probes is a powerful technique in analytical chemistry. Fluorescent chemosensors have wide-ranging applications in cell biology and analytical chemistry.
The majority of fluorescent sensors and biosensors do not undergo changes in emission spectral shape upon analyte binding and accordingly evince monochromatic intensity changes, rather than the dichromatic responses required for ratiometric sensing.
SUMMARY OF THE INVENTION
The present subject matter provides methods for converting monochromatic responses into dichromatic responses that enable ratiometric sensing. If the fluorescence emission spectrum changes shape in response to analyte binding such that the ratio of emission intensities at two appropriately chosen wavelengths reports on analyte concentration (dichromatic response), then ratiometric measurements can be used to monitor analyte concentrations. In embodiments, these methods are based on establishing non-geometrically modulated Forster resonance energy transfer (ngmFRET) between a monochromatic, chemoresponsive fluorophore (a directly responsive partner), and a second fluorophore that neither interacts directly with the ligand, nor is sensitive to ligand-mediated changes in its environment (an indirectly responsive partner). Biosensors that undergo ngmFRET (or altered ngmFRET) upon ligand binding are also provided herein, as well as compositions and devices comprising such biosensors.
Methods, compounds, and compositions provided herein overcome challenges regarding the design of biosensors that produce a ratiometric signal. For example, a biosensor that exhibits a monochromatic response (which does not produce a ratiometric signal) to ligand binding may be converted into a biosensor that produces a dichromatic/ratiometric signal. Moreover, the number of fluorophores that may be utilized in ratiometric biosensors is dramatically increased by the present subject matter. For example, fluorophores that typically do not show a dichromatic response to ligand binding (such as fluorescein and derivatives thereof) may be used together with an additional reporter group (such as another fluorophore) to produce a ratiometric signal. Also included are methods, compounds, and compositions relating to biosensors with multiple reporter groups that have improved ratiometric signals compared to other ratiometric biosensors (e.g., ratiometric biosensors having a single reporter group).
Traditional/conventional geometrically-modulated Fluorescence Resonance Energy Transfer (tgmFRET) is a physical phenomenon that was first described over 50 years ago. In tgmFRET, the transfer of excited state energy from a donor fluorophore to an acceptor fluorophore (i.e. energy transfer) is modulated by a ligand-binding event through changes in the distance and/or angle between the donor and acceptor fluorophores. tgmFRET
is manifested by opposing changes in the fluorescence emission intensities of the donor and acceptor fluorophores, respectively, in response to ligand binding. For instance, a decrease in distance results in a decrease of the donor fluorescence emission intensity and an increase in the acceptor fluorescence intensity, as energy is transferred from the former to the latter. A
ligand-mediated increase in the distance between the partners has the opposite effect (the fluorescence emission intensity of the donor increases, whereas that of the acceptor decreases). In tgmFRET, ligand-mediated modulation of fluorescence intensity arises from global changes in the entire system, and can occur only if both partners are present.

By contrast, in ngmFRET ligand-mediated modulation of fluorescence intensity arises from changes that are localized to the photophysics of the directly responsive fluorophore.
Unlike tgmFRET, ligand-mediated changes in fluorescence therefore occur also if only the directly responsive partner is present in isolation by itself. Although the entire ngmFRET
system comprising two partners is not required for evincing ligand-mediated changes in fluorescence emission intensity, the response of such a system is qualitatively changed or quantitatively enhanced over the responses of the isolated directly responsive partner (e.g.
converting a monochromatic into a dichromatic response, thereby enabling ratiometry).
Furthermore, unlike tgmFRET, the pattern of fluorescence intensity changes manifested by ligand binding in ngmFRET systems are not limited to opposing changes only.
Instead, in ngmFRET almost all combinations of emission intensity changes are possible:
opposing changes in the two partners, both partners increase, both decrease, one partner remains unchanged whereas the other increases or decreases. The majority of these responses evince changes that are unequal in magnitude and/or direction (i.e. increase, decrease), and accordingly are manifested as ligand-mediated changes in the ratio of the two fluorescence emission intensities. This versatility of ngmFRET system response patterns has great utility in the field of fluorescent biosensors.
The ligand-mediated alteration of the photophysics of the directly responsive partner includes changes to its spectral properties such as the shape of the excitation or emission spectra, and the ratio of radiative to non-radiative emission rates. The fluorescence emission intensity of the indirectly responsive partner in isolation does not change in response to ligand binding; its intensity changes only in the presence of a directly responsive partner in the complete ngmFRET system. In the field fluorescence spectroscopy, the term "quenching" has often been used loosely to refer to a decrease fluorescence emission intensity. However, as used herein, the term "quenching" strictly means a "change in the ratio of radiative to non-radiative emission rates" of a fluorophore.
Aspects of the present subject matter provide biosensors in which ngmFRET
occurs between two or more reporter groups (e.g., a donor fluorophore and an acceptor fluorophore) of the biosensor. For example, ngmFRET may change (e.g., increase or decrease) when ligand is bound to the biosensor and a donor fluorophore is contacted with radiation within its excitation wavelength. Effects from tgmFRET and ngmFRET may occur together and be combined into an overall ligand-mediated change in fluorescence emission intensity. In preferred embodiments, less than half or none of the change in overall ligand-mediated

3 change in fluorescence emission intensity is due to tgmFRET. In embodiments, most of the overall ligand-mediated change in fluorescence emission intensity change is not due to a change in the distance between the donor and acceptor fluorophore or as a result of a change in the orientation between the donor and acceptor fluorophore. In non-limiting examples, less than about 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or 0.5% of the change in overall ligand-mediated change in fluorescence emission intensity is due to tgmFRET.
In various embodiments, at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 99.99% of the ligand-mediated change in fluorescence emission intensity is due to ngmFRET. For example, the change in overall ligand-mediated change in fluorescence emission intensity comprises a spectral change (e.g., in the excitation or emission spectrum) and/or a change in the ratio of the radiative to non-radiative decay rates of one of the fluorophores (by itself and regardless of the presence of any other fluorophore/partner) upon ligand binding.
In some embodiments, ligand binding mediates spectral shifts in the absorption or emission spectrum of the directly responsive partner. In certain embodiments such changes are due at least in part to a switch between different excited states in the ligand-free and ligand-bound biosensor. The two excited states are associated with different transition dipoles. This class of changes is termed "dipole switching" herein. Non-limiting examples of biosensors that show dipole sensing include ttGGBP 17C=Badan-f3Zif Alexa532 and ttGGBP 182C =Acrylodan-f3Zif Alexa532.
In embodiments, the reporter groups include a directly responsive partner (which may be a donor fluorophore or an acceptor fluorophore) and an indirectly responsive partner (which may be a donor fluorophore or an acceptor fluorophore). Depending on context, a "directly responsive" partner is a fluorophore that responds to (i) ligand-induced protein conformational changes upon ligand binding to a ligand-binding protein; or (ii) ligand binding to the directly responsive partner itself. In some embodiments, the directly responsive partner comprises a fluorophore (i.e., it is a directly responsive fluorophore). In various embodiments, the directly responsive fluorophore exhibits a monochromatic or dichromatic spectral change, and/or a change in the ratio of radiative to non-radiative emission rates, upon ligand binding. In certain embodiments relating to ligand binding to the directly responsive partner itself, the directly responsive partner may be a fluorophore such as a fluorescent protein or a small molecule fluorescent compound. An "indirectly responsive"
partner is a fluorophore for which no change in emission spectra, excitation spectra, or

4 change in the ratio of radiative to non-radiative emission rates is caused by ligand binding in the absence of a directly responsive partner. In some embodiments, the indirectly responsive partner comprises a fluorophore (i.e., it is an indirectly responsive fluorophore). When paired with a directly responsive partner with which the indirectly responsive partner is a ngmFRET
donor or acceptor, the emission fluorescence intensity of the indirectly responsive partner changes due to a change in energy flow in the ngmFRET pathway upon ligand binding. See, e.g., FIG. 28.
ngmFRET Biosensors Provided herein are methods, compositions, biosensors, and devices comprising multiple reporter groups, e.g. a directly responsive fluorophore and an indirectly responsive fluorophore, between which ngmFRET occurs.
Aspects include a method of detecting a ligand in a sample, comprising contacting a biosensor with a ligand. The biosensor comprises a ligand-binding protein, a directly responsive fluorophore and an indirectly responsive fluorophore. The directly responsive and the indirectly responsive fluorophores are located at two distinct sites of the ligand-binding protein. In some embodiments, the directly responsive fluorophore is a donor fluorophore and the indirectly responsive fluorophore is an acceptor fluorophore.
Alternatively, the directly responsive fluorophore is an acceptor fluorophore and the indirectly responsive fluorophore is a donor fluorophore. The method includes contacting the biosensor with radiation comprising a wavelength within the excitation spectrum of the donor fluorophore.
When the biosensor is contacted with such radiation, a fluorescence property of the directly responsive fluorophore changes in response to ligand binding. This change in fluorescent property is independent of the indirectly responsive fluorophore, and occurs regardless of whether the indirectly responsive fluorophore is absent or present. The fluorescence properties of the indirectly responsive fluorophore do not change in response to ligand binding in the absence of the directly responsive fluorophore. When the biosensor is contacted with radiation comprising a wavelength within the excitation spectrum of the donor fluorophore, then (i) ngmFRET occurs between the directly responsive fluorophore and the indirectly responsive fluorophore; (ii) fluorescent light is emitted from the biosensor, and the light emitted from the biosensor comprises a combination of light emitted from the directly responsive fluorophore and light emitted from the indirectly responsive fluorophore; and (iii) the ratio of the fluorescence emission intensity emitted from the biosensor at each of two

5 distinct wavelengths changes in response to ligand binding. In various embodiments, the method further comprises measuring fluorescent light that is emitted from the directly responsive fluorophore and the indirectly responsive fluorophore, and calculating a ratiometric signal to detect the ligand in the sample.
The ratiometric signal (R1,2) comprises a quotient of two intensities, /xi and /x2, measured at two independent wavelengths, Xi and k2 and is calculated according to the following equation:
R1,2 = /Ai //A2 =
In various embodiments, the change in the fluorescent property of the directly responsive fluorophore comprises (i) a bathochromic or hypsochromic shift in the emission or excitation spectrum thereof; and/or (ii) a change in the ratio of radiative to non-radiative emission rates thereof.
In some embodiments, the directly responsive fluorophore is Badan and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and 1000nm (e.g., including a wavelength of about 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, or 475 nm), and wherein the indirectly responsive fluorophore is 5-iodoacetamidofluorescein (5-IAF) and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and 1000nm (e.g., including a wavelength of about 510, 511, 512, 513, 514, 515, 516,517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, or 530 nm).
In certain embodiments, the directly responsive fluorophore is Badan and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and 1000nm (e.g., including a wavelength of about 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, or 475 nm), and wherein the indirectly responsive fluorophore is A1exa532 and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and 1000nm (e.g., including a wavelength of about 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, or 570 nm).
In various embodiments, the directly responsive fluorophore is Pacific Blue and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and 1000nm (e.g., including a wavelength of about 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, or 465 nm), and wherein the

6 indirectly responsive fluorophore is 5-IAF and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and 1000nm (e.g., including a wavelength of about 510, 511, 512, 513, 514, 515, 516,517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, or 530 nm).
In some embodiments, the directly responsive fluorophore is Acrylodan and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and 1000nm (e.g., including a wavelength of about 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, or 475 nm), and wherein the indirectly responsive fluorophore is 5-IAF and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and 1000nm (e.g., including a wavelength of about 510, 511, 512, 513, 514, 515, 516,517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, or 530 nm).
In some embodiments, the directly responsive fluorophore is Acrylodan and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and 1000nm (e.g., including a wavelength of about 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490 nm), and wherein the indirectly responsive fluorophore is A1exa532 and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and 1000nm (e.g., including a wavelength of about 540, 541, 542,543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, or 560 nm).
In certain embodiments, the directly responsive fluorophore is 5-IAF and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and 1000nm (e.g., including a wavelength of about 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, or 465 nm), and wherein the indirectly responsive fluorophore is Pacific Blue and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and 1000nm (e.g., including a wavelength of about 510, 511, 512, 513, 514, 515, 516,517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, or 530 nm).
In various embodiments, the directly responsive fluorophore is Oregon Green and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and 1000nm (e.g., including a wavelength of about 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, or 465 nm), and wherein the indirectly responsive fluorophore is Pacific Blue and emission intensity is measured at a

7 wavelength or range of wavelengths between about 400 nm and 1000nm (e.g., including a wavelength of about 510, 511, 512, 513, 514, 515, 516,517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, or 530 nm).
In some embodiments, the directly responsive fluorophore is N-(Iodoacetaminoethyl)-1-naphthylamine-5-sulfonic acid (IAEDANS) and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and 1000nm (e.g., including a wavelength of about 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, or 475 nm), and wherein the indirectly responsive fluorophore is 5-IAF and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and 1000nm (e.g., including a wavelength of about 510, 511, 512, 513, 514, 515, 516,517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, or 530 nm).
In some embodiments, the directly responsive fluorophore is A1exa532 and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and 1000 nm (e.g. including a wavelength of about 530, 531, 532, 534, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, or 570 nm), and wherein the indirectly responsive fluorophore is Acrylodan and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and 1000 nm (e.g.
including 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 45, 496, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, or 510 nm).
In various embodiments, the directly responsive fluorophore is a yellow fluorescent protein and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and 1000nm (e.g., including a wavelength of about 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, or 540 nm), and wherein the indirectly responsive fluorophore is Acrylodan and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and 1000nm (e.g., including a wavelength of about 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, or 510 nm. In certain embodiments, the directly responsive fluorophore is a yellow fluorescent protein and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and 1000nm (e.g., including a wavelength of about 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533,

8 534, 535, 536, 537, 538, 539, or 540 nm), and wherein the indirectly responsive fluorophore is Pacific Blue and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and 1000nm (e.g., including a wavelength of about 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, or 465 nm).
In embodiments, the directly responsive fluorophore comprises a donor fluorophore and the indirectly responsive fluorophore comprises an acceptor fluorophore.
In some embodiments, the emission intensity of the donor fluorophore decreases and the emission intensity of the acceptor fluorophore increases upon ligand binding to the ligand-binding protein when the donor fluorophore is contacted with radiation within the excitation spectrum of the donor fluorophore. In some embodiments, the emission intensity of the donor fluorophore increases and the emission intensity of the acceptor fluorophore decreases upon ligand binding to the ligand-binding protein when the donor fluorophore is contacted with radiation within the excitation spectrum of the donor fluorophore. In some embodiments, the emission intensities of the donor fluorophore and the acceptor fluorophore both decrease upon ligand binding to the ligand-binding protein when the donor fluorophore is contacted with radiation within the excitation spectrum of the donor fluorophore. In some embodiments, the emission intensity of the donor fluorophore decreases and the emission intensity of the acceptor fluorophore increases, decreases, or remains about the same upon ligand binding to the ligand-binding protein when the donor fluorophore is contacted with radiation within the excitation spectrum of the donor fluorophore. In some embodiments, the emission intensity of the donor fluorophore increases, decreases, or remains about the same and the emission intensity of the acceptor fluorophore decreases upon ligand binding to the ligand-binding protein when the donor fluorophore is contacted with radiation within the excitation spectrum of the donor fluorophore. In some embodiments, the emission intensities of the donor fluorophore and the acceptor fluorophore both increase upon ligand binding to the ligand-binding protein when the donor fluorophore is contacted with radiation within the excitation spectrum of the donor fluorophore. In some embodiments, the emission intensity of the donor fluorophore increases, decreases, or remains about the same and the emission intensity of the acceptor fluorophore increases upon ligand binding to the ligand-binding protein when the donor fluorophore is contacted with radiation within the excitation spectrum of the donor fluorophore. In some embodiments, the emission intensity of the donor fluorophore increases and the emission intensity of the acceptor fluorophore increases,

9 decreases, or remains about the same upon ligand binding to the ligand-binding protein when the donor fluorophore is contacted with radiation within the excitation spectrum of the donor fluorophore.
In embodiments the directly responsive fluorophore comprises an acceptor fluorophore and the indirectly responsive fluorophore comprises a donor fluorophore. In some embodiments, the emission intensity of the donor fluorophore decreases and the emission intensity of the acceptor fluorophore increases, decreases, or remains about the same upon ligand binding to the ligand-binding protein when the donor fluorophore is contacted with radiation within the excitation spectrum of the donor fluorophore. In some embodiments, the emission intensity of the donor fluorophore increases and the emission intensity of the acceptor fluorophore increases, decreases, or remains about the same upon ligand binding to the ligand-binding protein when the donor fluorophore is contacted with radiation within the excitation spectrum of the donor fluorophore. In some embodiments, the emission intensity of the donor fluorophore remains about the same and the emission intensity of the acceptor fluorophore decreases upon ligand binding to the ligand-binding protein when the donor fluorophore is contacted with radiation within the excitation spectrum of the donor fluorophore. In some embodiments, the emission intensity of the donor fluorophore decreases and the emission intensity of the acceptor fluorophore increases, decreases, or remains about the same upon ligand binding to the ligand-binding protein when the donor fluorophore is contacted with radiation within the excitation spectrum of the donor fluorophore. In some embodiments, the emission intensity of the donor fluorophore increases and the emission intensity of the acceptor fluorophore increases, decreases, or remains about the same upon ligand binding to the ligand-binding protein when the donor fluorophore is contacted with radiation within the excitation spectrum of the donor fluorophore. In some embodiments, the emission intensity of the donor fluorophore remains about the same and the emission intensity of the acceptor fluorophore increases upon ligand binding to the ligand-binding protein when the donor fluorophore is contacted with radiation within the excitation spectrum of the donor fluorophore. In some embodiments, the emission intensity of the donor fluorophore decreases and the emission intensity of the acceptor fluorophore increases upon ligand binding to the ligand-binding protein when the donor fluorophore is contacted with radiation within the excitation spectrum of the donor fluorophore. In some embodiments, the emission intensity of the donor fluorophore increases and the emission intensity of the acceptor fluorophore remains about the same, increases, or decreases upon ligand binding to the ligand-binding protein when the donor fluorophore is contacted with radiation within the excitation spectrum of the donor fluorophore.
In instances in which an emission intensity increases, the increase may be, e.g., between about 0.1% to 10%, 10% to 50%, or 50% to 100%, or at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 50%, 75%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold. In instances in which an emission intensity decreases, the decrease may be, e.g., a decrease of between about at least about 0.1% to

10%, 10% to 50%, or 50% to 100%, or at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%. In various embodiments in which both the emission intensity of the donor fluorophore and the acceptor fluorophore increases, then the increases are not equal. In certain embodiments in which both the emission intensity of the donor fluorophore and the acceptor fluorophore decreases, then the decreases are not equal.
In various embodiments, the ligand-binding protein comprises the directly responsive fluorophore. For example, the directly responsive fluorophore is formed by an autocatalytic cyclization of an oligopeptide within the ligand-binding protein. In some embodiments, the oligopeptide is located within an interior a helix. In certain embodiments, the oligopeptide comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive residues. In embodiments, the directly responsive fluorophore is formed by an autocatalytic cyclization of a tipeptide located in an interior a helix of the ligand-binding protein. In various embodiments, ligand-binding protein comprises a yellow fluorescent protein (YFP), i.e. the YFP binds to ligand such as a halide anion.
In some embodiments, ligand binding causes a change in signaling by the directly responsive fluorophore.
Also provided is a method of detecting a ligand in a sample, comprising contacting a biosensor with a ligand, wherein the biosensor comprises an amino acid or polypeptide, a directly responsive fluorophore and an indirectly responsive fluorophore. The directly responsive and the indirectly responsive fluorophores are located at two distinct sites of the amino acid or polypeptide, and the directly responsive fluorophore is chemoresponsive. The method may further comprise contacting the biosensor with radiation comprising a wavelength within the excitation spectrum of the donor fluorophore, wherein (i) a fluorescence property of the directly responsive fluorophore changes in response to ligand binding in the absence or presence of the indirectly responsive fluorophore;
(ii) a

11

12 fluorescence property of the indirectly responsive fluorophore does not change in response to ligand binding in the absence of the directly responsive fluorophore; (iii) ngmFRET occurs between the directly responsive fluorophore and the indirectly responsive fluorophore; (iv) fluorescent light is emitted from the biosensor, wherein the light emitted from the biosensor comprises a combination of light emitted from the directly responsive fluorophore and light emitted from the indirectly responsive fluorophore; and (v) the ratio of the fluorescence emission intensity emitted from the biosensor at each of two distinct wavelengths changes in response to ligand binding. The method may also include measuring fluorescent light that is emitted from the directly responsive fluorophore and the indirectly responsive fluorophore and calculating a ratiometric signal, to detect the ligand in the sample. The ratiometric signal (R1,2) comprises a quotient of two intensities, Ai and A2, measured at two independent wavelengths, Xi and k2 and is calculated according to the following equation:
R1,2 = '2i/'22 =
As used herein, a "chemoresponsive" fluorophore is a fluorophore to which ligand binds, wherein ligand binding causes a change in signaling by the fluorophore.
As used herein, "signaling" refers to the emission of energy (which may be referred to as a "signal") by one or more reporter groups. In various implementations, the signal comprises electromagnetic radiation such as a light. In some embodiments, the signal is detected as a complete emission spectrum (or spectra) or a portion (or portions) thereof. For example, a signal may comprise emitted light at a particular wavelength or wavelengths, or range(s) of wavelengths. In some embodiments, a change in signaling comprises a spectral change (e.g., a spectral shift and/or change in intensity). In some embodiments, a change in signaling comprises a dichromatic shift or a monochromatic fluorescence intensity change.
In some embodiments, the directly responsive fluorophore is a donor fluorophore and the indirectly responsive fluorophore is an acceptor fluorophore.
Alternatively, the directly responsive fluorophore is an acceptor fluorophore and the indirectly responsive fluorophore is a donor fluorophore.
In various embodiments, the change in the fluorescent property of the directly responsive fluorophore comprises (i) a bathochromic or hypsochromic shift in the emission or excitation spectrum thereof; and/or (ii) a change in the ratio of radiative to non-radiative emission rates thereof.

In some embodiments, the directly responsive fluorophore is 5-IAF and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and 1000nm (e.g., including a wavelength of about 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, or 470 nm), and wherein the indirectly responsive fluorophore is Acrylodan and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and 1000nm (e.g., including a wavelength of about 510, 511, 512, 513, 514, 515, 516,517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, or 530 nm).
In certain embodiments, the directly responsive fluorophore is 5-IAF and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and 1000nm (e.g., including a wavelength of about 510, 511, 512, 513, 514, 515, 516,517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, or 530 nm), and wherein the indirectly responsive fluorophore is Pacific Blue and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and 1000nm (e.g., including a wavelength of about 445, 446, 447,448, 449, 450, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, nm).
Any amino acid or polypeptide may be used to link the chemoresponsive directly responsive fluorophore with the indirectly responsive fluorophore, provided the two fluorophores are close enough for ngmFRET to occur. Suitable distances may be determined in part by the distance-dependence of the energy transfer between a given donor-acceptor pair (see, e.g, J.R. Lakowicz, 2006, Principles of Fluorescence Spectroscopy, Springer, incorporated herein by reference). In various embodiments, the amino acid or polypeptide comprises 1 amino acid, or a stretch of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 500, 750, or 1000 amino acids. In some embodiments, the amino acid or polypeptide comprises at least 1, 2, or 3 thiol groups; at least 1, 2, or 3 cysteines that each comprise a sulfhydryl group; at least 1, 2, or 3 primary amine groups; or at least 1, 2, or 3 lysines that each comprise a primary amine. In certain embodiments, the polypeptide comprise two cysteines, and there is no disulfide bond between the two cysteines. In some embodiments there is no disulfide bond between any pair of cysteines within the amino acid sequence of the polypeptide.
In a non-limiting example, the polypeptide comprises a stretch of at least 50, 60, 70, 80, 90, or 100 amino acids in a sequence that is at least about 85%, 90%, 95%, or 99%
identical to the amino acid sequence of ecTRX (SEQ ID NO: 151). In some embodiments,

13 the polypeptide comprises a mutant of ecTRX comprising a D3X, K4X, K19X, D27X, K37X, K53X, K58X, K70X, R74X, K83X, K91X, K97X, or K101X mutation, or any combination thereof, wherein X is any amino acid, and wherein each ecTRX amino acid position is numbered as in SEQ ID NO: 151. In certain embodiments, the polypeptide comprises a mutant of ecTRX comprising a D3A, K4R, K4Q, K19R, K19Q, D27A, K37R, K53M, K53R, K58M, K7OR, R74C, K83R, K91R, K97R, or K101R mutation, or any combination thereof, wherein each ecTRX amino acid position is numbered as in SEQ ID NO: 151. In various embodiments, the polypeptide comprises a mutant of ecTRX that does not comprise a lysine.
In certain embodiments, the polypeptide comprises amino acids in the sequence of any one of SEQ ID NOS: 69-86 or 151.
In certain embodiments, the polypeptide further comprises a hexahistidine tag.
In some embodiments, the ligand comprises a hydrogen ion. For example, the biosensor for pH, wherein the directly responsive fluorophore is pH-sensitive.
In various embodiments, the fully excited emission intensity of the directly responsive fluorophore is different at a pH less than about 7.0 (e.g. 6.9, 6.8, 67, 6.6, 6.5, 6.4, 6.3, 6.2, 6.1, or 6.0), or about 4.0 to 10.0, compared to a pH of about 7.3, 7.4, 7.5, 7.6, or 7.7.
In various embodiments, the directly responsive fluorophore comprises a pH-sensitive fluorophore comprising fluorescein or a derivative thereof. In embodiments, the directly responsive fluorophore transitions from a monoanion to a dianion at a pH that is less than 7.0 in an aqueous solution.
In certain embodiments, the indirectly responsive fluorophore is attached to the ligand-binding protein via a covalent bond. Various approaches for attaching reporter groups such as directly and indirectly responsive fluorophores to an amino acid or a polypeptide such as a ligand-binding protein are described herein. In some embodiments, the covalent bond comprises a disulfide bond, a thioester bond, a thioether bond, an ester bond, an amide bond, or a bond that has been formed by a click reaction.
In some embodiments, the indirectly responsive fluorophore is attached to the ligand-binding protein via a non-covalent bond. In certain embodiments, the indirectly responsive fluorophore is attached to a cysteine or a lysine of the protein.
In various embodiments, the indirectly responsive fluorophore is attached to the N-terminus or the C-terminus of the protein. In some embodiments, the indirectly responsive fluorophore is attached to the N-terminus or the C-terminus of the protein via a fluorophore attachment motif.

14 In some embodiments, fluorophore attachment motif comprises an amino acid or polypeptide. Various embodiments may be used to link a fluorophore with a ligand-binding protein. In some embodiments, the amino acid or polypeptide comprises 1 amino acid, or a stretch of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 500, 750, or 1000 amino acids. In a non-limiting example, the polypeptide comprises amino acids in the sequence of PZif (SEQ ID NO: 42). In another non-limiting example, the polypeptide comprises a stretch of at least 50, 60, 70, 80, 90, or 100 amino acids in a sequence that is at least about 85%, 90%, 95%, or 99% identical to the amino acid sequence of E. coli thioredoxin (ecTRX; SEQ ID NO: 151).
In some embodiments, the directly responsive fluorophore is attached to the ligand-binding protein via a covalent bond. In various embodiments, the covalent bond comprises a disulfide bond, a thioester bond, a thioether bond, an ester bond, an amide bond, or a bond that has been formed by a click reaction. In directly responsive fluorophore is attached to a cysteine or a lysine of the protein.
In various embodiments, if the acceptor fluorophore comprises palladium, platinum, ruthenium, or osmium, then the acceptor fluorophore is not attached to the amino group of the N-terminus of the ligand-binding protein. In some embodiments, the acceptor fluorophore does not comprise [Itu(bpy)3j2"-, iltu(Ph2phen)3i2+, [Itu(bpy)2(dobpy)]2+, or [Ru(lopy)2(phen-ITC)]24, where bpy is 2,2!--bipyridine, phen is 1,10-phenanthroline, debpy is 4,4'-dicarboxy-2,2'-bipyridine, and ITC is isothiocyanate. In certain embodiments, the biosensor does not comprise an E. coli glutamine-binding protein with Acrylodan attached to 179C. In some embodiiments, the biosensor does not comprise E. coli glucose-binding protein with Acrylodan attached to 255C.
In some embodiments, an overlap of the emission spectrum of the donor fluorophore and the excitation spectrum of the acceptor fluorophore increases upon ligand binding. In certain embodiments, the directly responsive fluorophore comprises the donor fluorophore, and the increase results from a bathochromic shift in the emission spectrum of the donor fluorophore. Alternatively, the directly responsive fluorophore comprises the acceptor fluorophore, and the increase results from a hypsochromic shift in the excitation spectrum of the acceptor fluorophore.
In various embodiments, an overlap of the emission spectrum of the donor fluorophore and the excitation spectrum of the acceptor fluorophore decreases upon ligand binding. In some embodiments, the directly responsive fluorophore comprises the donor fluorophore, and the decrease results from a hypsochromic shift in the emission spectrum of the donor fluorophore. In certain embodiments, the directly responsive fluorophore comprises the acceptor fluorophore, and the decrease results from a bathochromic shift in the excitation spectrum of the acceptor fluorophore.
In some embodiments, the directly responsive fluorophore has a monochromatic spectral change upon ligand binding. Alternatively, the directly responsive fluorophore has a dichromatic spectral change upon ligand binding.
In certain embodiments, the emission intensity of the donor fluorophore and/or the acceptor fluorophore increases in two phases as ligand concentration increases.
In various embodiments, the ratio of radiative to non-radiative emission or intensity of the directly responsive fluorophore increases by at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 50%, 75%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold upon ligand binding to the ligand-binding protein.
Alternatively, the ratio of radiative to non-radiative emission or intensity of the directly responsive fluorophore decreases by at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 50%, 75%, 90%, 95%, or 99% upon ligand binding to the ligand-binding protein.
In embodiments, the directly responsive fluorophore and the indirectly responsive fluorophore are not a naphthalene derivative. In some embodiments, the directly responsive fluorophore and the indirectly responsive fluorophore are not Prodan, Acrylodan, or Badan.
In certain embodiments, the directly responsive fluorophore is not a naphthalene derivative.
In some embodiments, the directly responsive fluorophore is not Prodan, Acrylodan, or Badan.
In various embodiments, the directly responsive fluorophore comprises xanthene, a xanthene derivative, fluorescein, a fluorescein derivative, coumarin, a coumarin derivative, cyanine, a cyanine derivative, rhodamine, a rhodamine derivative, phenoxazine, a phenoxazine derivative, squaraine, a squaraine derivative, coumarin, a coumarin derivative, oxadiazole, an oxadiazole derivative, anthracene, an anthracene derivative, a boradiazaindacine (BOD1PY) family fluorophore, pyrene, a pyrene derivative, acridine, an acridine derivative, arylmethine, an arylmethine derivative, tetrapynole, or a tetrapynole derivative. In some embodiments, the directly responsive fluorophore comprises fluorescein or a derivative thereof.
In some embodiments, the directly responsive fluorophore and/or the indirectly responsive fluorophore comprises a fluorescent protein. In various embodiments, the directly responsive fluorophore and/or the indirectly responsive fluorophore comprises an organic compound having a molecular weight less than about 2000 Da (e.g., 5-iodoacetamidofluorescein (5-IAF) or 6-iodoacetamidofluorescein (6-IAF), rhodamine, Oregon Green, eosin, Texas Red, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, merocyanine, Badan, Acrylodan, IAEDANS, comprising 3-cyano-7-hydroxycoumarin, hydroxycoumarin-3-carboxylic acid, 6,8-difluoro-7-hydroxy- 4-methylcoumarin, or 7-amino-4-methylcoumarin, pyridyloxazole, nitrobenzoxadiazole, benzoxadiazole, DRAQ5, DRAQ7, or CyTRAK Orange, cascade blue, Nile red, Nile blue, cresyl violet, oxazine 170, proflavin, acridine orange, acridine yellow, auramine, crystal violet, malachite green, porphin, phthalocyanine, bilirubin, pyrene, N,Nt-dimethyl-N-(iodoacety1)-N'-(7-nitrobenz-2-ox- a-1,3-diazol-4-ypethylenediamide (NBD), N-((2-(iodoacetoxy)ethyl)-N-methy- 1)amino-7-nitrobenz-2-oxa-1,3-diazole (NBDE), JPW4039, JPW4042, JPW4045, Pacific Blue, CPM, N,Nt-Dimethyl-N-(Iodoacety1)-N'-(7-Nitrobenz-2-Oxa-1,3-Diazol-4-y1)Ethylenediamine (IANBD), 7-diethylamino-3-(4'-maleimidylpheny1)-4-methylcoumarin (CPM), BODIPY
499, BODIPY 507/545, BODIPY 499/508, Alexa 432, A1exa488, A1exa532, A1exa546, Cy5, or 1-(2-maleimidylethyl)-4-(5-(4-methoxyphenypoxazol-2-yppyridinium methanesulfonate (PyMPO maleimide) (PyMPO)). Numerous combinations of directly responsive fluorophores and indirectly responsive fluorophores are possible. For example, in various non-limiting examples, (a) the donor fluorophore comprises Pacific Blue and the acceptor fluorophore comprises 5-IAF or 6-iodoacetamidofluorescein (6-IAF); (b) the donor fluorophore comprises Pacific Blue and the acceptor fluorophore comprises Oregon Green;
(c) the donor fluorophore comprises IAEDANS and the acceptor fluorophore comprises 5-IAF or 6-IAF; (d) the donor fluorophore comprises acrylodan and the acceptor fluorophore comprises Alexa532; (e) the donor fluorophore comprises acrylodan and the acceptor fluorophore comprises 5-IAF or 6-IAF; (f) the donor fluorophore comprises acrylodan and the acceptor fluorophore comprises Pacific Blue or YFP; (g) the donor fluorophore comprises 5-IAF or 6-IAF and the acceptor fluorophore comprises Pacific Blue; (h) the donor fluorophore comprises badan and the acceptor fluorophore comprises 5-IAF or 6-IAF; or (i) the donor fluorophore comprises badan and the acceptor fluorophore comprises Alexa532.
Any of the ligand-binding proteins disclosed herein, as well as others, may be included in the biosensors and methods that are provided. In some embodiments, the ligand-binding protein is selected from the group consisting of a glucose-galactose binding protein (GGBP), a glucose-binding protein, a urea-binding protein (UBP), a lactate-binding protein (LacBP), a calcium-binding protein, a calcium-bicarbonate binding protein (BicarbBP), and an iron-bicarbonate binding protein (FeBP).
Aspects include a biosensor for a ligand comprising a ligand-binding protein, a directly responsive fluorophore and an indirectly responsive fluorophore, the directly responsive and the indirectly responsive fluorophores being located at two distinct sites of the ligand-binding-protein, wherein (i) the directly responsive fluorophore is a donor fluorophore and the indirectly responsive fluorophore is an acceptor fluorophore; or (ii) the directly responsive fluorophore is an acceptor fluorophore and the indirectly responsive fluorophore is an donor fluorophore, and wherein if the acceptor fluorophore comprises ruthenium or osmium, then the acceptor fluorophore is not attached to the amino group of the N-terminus of the ligand-binding protein.
In some embodiments, the ligand-binding protein comprises the directly responsive fluorophore. In certain embodiments, the directly responsive fluorophore is formed by an autocatalytic cyclization of an oligopeptide within the ligand-binding protein.
In various embodiments, the ligand-binding protein comprises a Yellow Fluorescent Protein (YFP; SEQ ID NO: 149) or a fluorescent mutant thereof, and the ligand comprises a halide anion. For example, the halide anion comprises a fluoride (F), chloride (CF), a bromide (BO, an iodide (F), an astatide (At-) anion, or an ununseptide (Ts) anion. In some embodiments, the mutant comprises a mutation that alters the interaction of the mutant with a bound halide anion compared to YFP. In certain embodiments, the mutant comprises a mutation that alters the affinity and/or specificity of the mutant for a halide anion compared to YFP. In various embodiments, the ligand-binding protein comprises 1, 2, 3, 4, or 5 halide anion binding sites.
In some embodiments, at least one amino acid of the YFP or the fluorescent mutant thereof has been substituted with a cysteine. For example, the cysteine is within a first J3-strand (Pi), a second J3-strand (f32), a third J3-strand (f33), a fourth J3-strand (f34), a fifth J3-strand (135), a sixth J3-strand (P), a seventh J3-strand (P), an eighth J3-strand (f38), a ninth J3-strand (P), a tenth J3-strand (010), or an eleventh J3-strand (01i) of the YFP or the fluorescent mutant thereof. In certain embodiments, the ligand-binding protein comprises one or more of the following substitutions: E17X, E32X, T43X, F64X, G65X, L68X, Q69X, A72X, H77X, K79X, R80X, E95X, R109X, R122X, D133X, H148X, N149X, V163X, N164X, D173X, Y182X, Q183X, Y203X, Q204X, L221X, and H231X, wherein X is any amino acid, a conservative substitution, or a cysteine, wherein each YFP amino acid position is numbered as in SEQ ID NO: 150. In non-limiting examples, the ligand-binding protein comprises one or more of the following substitutions: F64L, G65T, L68V, Q69T, A72S, K79R, R80Q, H148Q, H148G, V163A, H231L, H148Q, or Q183A, wherein each YFP amino acid position is numbered as in SEQ ID NO: 150. In various embodiments, the ligand-binding protein comprises an R at the 96 position, a Y at the 203 position, a S at the 205 position, and an E at the 222 position, wherein each YFP amino acid position is numbered as in SEQ
ID NO: 150.
In various embodiments, ligand binding causes a change in signaling by the directly responsive fluorophore. In embodiments, the ligand-binding protein comprises a mutation compared to a naturally occurring protein. For example, at least one amino acid of the ligand-binding protein has been substituted with a cysteine. In some embodiments, the ligand-binding protein comprises a mutant of a microbial ligand-binding protein. In certain embodiments, the ligand-binding protein comprises a mutant of a microbial periplasmic ligand-binding protein.
In certain embodiments, the ligand comprises glucose, galactose, lactose, arabinose, ribose, maltose, lactate, urea, bicarbonate, phosphate, sulfate, chloride, fluoride, iodide, astatide, ununseptide, bromide, calcium, a hydrogen ion, a dipeptide, histidine, glutamine, glutamate, aspartate, or iron.
In some embodiments, the ligand-binding protein comprises a GGBP. For example, the GGBP comprises or comprises a mutant of: an Escherichia sp. GGBP; a Thermoanaerobacter sp. GGBP; a Clostridium sp. GGBP; a Salmonella sp. GGBP; a Caldicellulosiruptor sp. GGBP; a Paenibacillus sp. GGBP; a Butyrivibrio sp.
GGBP; a Roseburia sp. GGBP; a Faecalibacterium sp. GGBP; an Erysipelothrix sp. GGBP;
or an Eubacterium sp. GGBP.
In some embodiments, the ligand-binding protein comprises a UBP. For example, the UBP comprises or comprises a mutant of: an Marinomas sp. UBP; a Marinobacter sp. UBP;
a Bacillus sp. UBP; a Desulfotomaculum sp. UBP; a Geobacillus sp. UBP; a Clostridium sp.
UBP; a Caldicellulosiruptor sp. UBP; a Thermocrinis sp. UBP; a Synechoccus sp UBP; a Paenibacillus sp. UBP; or a Thermosynechococcus sp UBP.
In some embodiments, the ligand-binding protein comprises a GBP. For example, the GBP comprises or comprises a mutant of: an Thermus sp GBP; a Deinococcus sp.
GBP; a Thermotoga sp. GBP; a Kosmotoga sp. GBP; a Bacillus sp. GBP; a Staphylothermus sp.
GBP; or an Arthrobacter sp. GBP.

In some embodiments, the ligand-binding protein comprises a LacBP. For example, the LacBP comprises or comprises a mutant of: a Thermus sp. LacBP; a Thioalkalivibrio sp.
LacBP; a Roseobacter sp. LacBP; a Marinobacter sp. LacBP; a Anaeromyxobacter sp.
LacBP; a Pseudomonas sp. LacBP; a Rhodobacter sp. LacBP;, a Flexistipes sp.
LacBP; or a Thermanaerovibrio sp. LacBP.
In some embodiments, the ligand-binding protein comprises a calcium-binding protein or a BicarbBP. For example, the ligand-binding protein comprises or comprises a mutant of: a Synechocystis sp. BicarbBP; a Thermosynechococcus sp. BicarbBP; a Chroococcidiopsis sp. BicarbBP; a Calothrix sp. BicarbBP; a Anabaena sp.
BicarbBP; or a Chamaesiphon sp. BicarbBP.
In some embodiments, the ligand-binding protein comprises a FeBP. For example, the ligand-binding protein comprises or comprises a mutant of: a Mannheimia sp. FeBP; an Exiguobacterium sp. FeBP; a Thermosynechococcus sp FeBP; a Candidatus Nitrospira sp.
FeBP; a Thermus sp. FeBP; a Meiothermus sp. FeBP; a Salinibacter sp. FeBP; or a Halorubrum sp. FeBP.
Also provide is a biosensor for a ligand comprising an amino acid or a polypeptide, a directly responsive fluorophore and an indirectly responsive fluorophore, the directly responsive and the indirectly responsive fluorophores being located at two distinct sites of the amino acid or polypeptide, wherein the directly responsive fluorophore is chemoresponsive, and wherein (i) the directly responsive fluorophore is a donor fluorophore and the indirectly responsive fluorophore is an acceptor fluorophore; or (ii) the directly responsive fluorophore is an acceptor fluorophore and the indirectly responsive fluorophore is an donor fluorophore.
As noted above, any amino acid or polypeptide may be used to link the chemoresponsive directly responsive fluorophore with the indirectly responsive fluorophore, provided the two fluorophores are close enough for ngmFRET to occur. In some embodiments, the amino acid or polypeptide comprises 1 amino acid, or a stretch of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 500, 750, or 1000 amino acids.
In some embodiments, the polypeptide comprises a stretch of at least 50, 60, 70, 80, 90, or 100 amino acids in a sequence that is at least about 85%, 90%, 95%, or 99% identical to the amino acid sequence of ecTRX (SEQ ID NO: 151). In certain embodiments, the polypeptide comprises a mutant of ecTRX comprising a D3X, K4X, K1 9X, D27X, K37X, K53X, K58X, K70X, R74X, K83X, K91X, K97X, or K101X mutation, or any combination thereof, wherein X is any amino acid, and wherein each ecTRX amino acid position is numbered as in SEQ ID NO: 151. In some embodiments, the polypeptide comprises a mutant of ecTRX comprising a D3A, K4R, K4Q, K19R, K19Q, D27A, K37R, K53M, K53R, K58M, K7OR, R74C, K83R, K91R, K97R, or K101R mutation, or any combination thereof, wherein each ecTRX amino acid position is numbered as in SEQ ID NO: 151. In some embodiments, the polypeptide comprises a mutant of ecTRX that does not comprise a lysine.
In various embodiments, the biosensor comprises amino acids in the sequence of any one of SEQ ID
NOS: 69-86 or 151.
In some embodiments, the polypeptide further comprises a hexahistidine tag.
In certain embodiments, the amino acid or polypeptide comprises at least 1, 2, or 3 thiol groups; at least 1, 2, or 3 cysteines that each comprise a sulfhydryl group; at least 1, 2, or 3 primary amine groups; or at least 1, 2, or 3 lysines that each comprise a primary amine.
In some embodiments, there is no disulfide bond between cysteines within the amino acid sequence of the polypeptide.
In various embodiments, the ligand comprises a hydrogen ion. In some embodiments, the biosensor is a biosensor for pH, wherein the directly responsive fluorophore is pH-sensitive. In certain embodiments, the fully excited emission intensity of the directly responsive fluorophore is different at a pH less than about 7.0 compared to a pH of 7.5. In some embodiments, the directly responsive fluorophore comprises a pH-sensitive fluorophore comprising fluorescein or a derivative thereof. In some embodiments, the directly responsive fluorophore transitions from a monoanion to a dianion at a pH that is less than 7.0 in an aqueous solution.
In some embodiments, the directly responsive fluorophore is attached to the ligand-binding protein, the amino acid, or the polypeptide via a covalent bond. In some embodiments, the covalent bond comprises a disulfide bond, a thioester bond, a thioether bond, an ester bond, an amide bond, or a bond that has been formed by a click reaction. In certain embodiments, the directly responsive fluorophore is attached to a cysteine or a lysine of the protein.
In various embodiments, the indirectly responsive fluorophore is attached to the N-terminus or the C-terminus of the protein. In some embodiments, the indirectly responsive fluorophore is attached to the N-terminus or the C-terminus of the protein via a fluorophore attachment motif. In some embodiments, the fluorophore attachment motif comprises an amino acid or a polypeptide. In certain embodiments, the polypeptide comprises amino acids in the sequence of PZif (SEQ ID NO: 42). In various embodiments, polypeptide comprises a stretch of at least 50, 60, 70, 80, 90, or 100 amino acids in a sequence that is at least about 85%, 90%, 95%, or 99% identical to the amino acid sequence of E. coli thioredoxin (ecTRX;
SEQ ID NO: 151).
In certain embodiments, the indirectly responsive fluorophore is attached to the ligand-binding protein via a covalent bond. In some embodiments, the covalent bond comprises a disulfide bond, a thioester bond, a thioether bond, an ester bond, an amide bond, or a bond that has been formed by a click reaction. In various embodiments, the indirectly responsive fluorophore is attached to a cysteine or a lysine of the protein.
Aspects of the present subject matter further provide a method for assaying the level of a ligand in a subject, comprising contacting a biosensor with a biological sample from the subject. Non-limiting examples of ligands include glucose, galactose, lactose, arabinose, ribose, maltose, lactate, urea, bicarbonate, phosphate, sulfate, chloride, fluoride, iodide, astatide, ununseptide, bromide, calcium, a hydrogen ion, a dipeptide, histidine, glutamine, glutamate, aspartate, and iron.
In some embodiments, the subject has or is suspected of having abnormal kidney function, abnormal adrenal gland function, diabetes, hypochloremia, bromism, hypothyroidism, hyperthyroidism, cretinism, depression, fatigue, obesity, a low basal body temperature, a goiter, a fibrocystic breast change, lactic acidosis, septic shock, carbon monoxide poisoning, asthma, a lung disease, respiratory insufficiency, Chronic Obstructive Pulmonary Disease (COPD), regional hypoperfusion, ischemia, severe anemia, cardiac arrest, heart failure, a tissue injury, thrombosis, or a metabolic disorder, diarrhea, shock, ethylene glycol poisoning, methanol poisoning, diabetic ketoacidosis, hypertension, Cushing syndrome, liver failure, cancer, or an infection.
In various embodiments, the biological sample comprises sweat, tear fluid, blood, serum, plasma, interstitial fluid, amniotic fluid, sputum, gastric lavage, skin oil, milk, fecal matter, emesis, bile, saliva, urine, mucous, semen, lymph, spinal fluid, synovial fluid, a cell lysate, venom, hemolymph, or a fluid obtained from a plant.
Also provided is a method for assaying the level of ligand in an environmental sample, comprising contacting a biosensor with the environmental sample. In some embodiments, the environmental sample is from an environmental site that is suspected of being polluted. In some embodiments, the environmental sample has been obtained or provided from an environmental substance, fluid, or surface. In various embodiments, the environmental substance comprises (a) rock, soil, clay, sand, a meteorite, an asteroid, dust, plastic, metal, a mineral, a fossil, a sediment, or wood; (b) the environmental surface comprises the surface of a satellite, a bike, a rocket, an automobile, a truck, a motorcycle, a yacht, a bus, or a plane, a tank, an armored personnel carrier, a transport truck, a jeep, a mobile artillery unit, a mobile antiaircraft unit, a minesweeper, a Mine-Resistant Ambush Protected (MRAP) vehicle, a lightweight tactical all-terrain vehicle, a high mobility multipurpose wheeled vehicle, a mobile multiple rocket launch system, an amphibious landing vehicle, a ship, a hovercraft, a submarine, a transport plane, a fighter jet, a helicopter, a rocket, or an Unmanned Arial Vehicle, a drone, a robot, a building, furniture, or an organism; or (c) the environmental fluid comprises marine water, well water, drinking well water, water at the bottom of well dug for petroleum extraction or exploration, melted ice water, pond water, aquarium water, pool water, lake water, mud, stream water, river water, brook water, waste water, treated waste water, reservoir water, rain water, or ground water.
Aspects of the present subject matter further provide a method for monitoring the level of a ligand, comprising periodically continuously detecting the level of the ligand, wherein detecting the level of the ligand comprises (a) providing or obtaining a sample; (b) contacting the sample with a biosensor for the ligand; and (c) detecting a signal produced by the biosensor. In some embodiments, the sample is provided or obtained from a subject or from a culture of microbial cells.
Additional embodiments and methods for detecting the presence and/or amount of a ligand are disclosed herein.
Aspects of the present subject matter also provide a method for constructing a biosensor, comprising: (a) providing a ligand-binding protein; (b) identifying at least one putative allosteric, endosteric, or peristeric site of the ligand-binding based a structure of the ligand-binding protein; (c) mutating the ligand-binding protein to substitute an amino acid at the at least one putative allosteric, endosteric, or peristeric site of the second protein with a cysteine; (d) conjugating a donor fluorophore or an acceptor fluorophore to the cysteine to produce single labeled biosensor; (e) detecting whether there is a spectral shift or change in emission intensity of the single labeled biosensor upon ligand binding when the donor fluorophore or the acceptor fluorophore is fully excited; and (f) if a spectral shift or change in emission intensity is detected in (e), attaching a donor fluorophore to the second protein if an acceptor fluorophore is attached to the cysteine, and attaching an acceptor fluorophore to the second protein if an acceptor fluorophore is attached to the cysteine.

In various embodiments, the ligand-binding protein has been identified by (i) selecting a first protein having a known amino acid sequence (seed sequence), wherein the first protein is known to bind a ligand; (ii) identifying a second protein having an amino acid sequence (hit sequence) with at least 15% sequence identity to the seed sequence; (iii) aligning the seed amino acid sequence and the hit sequence, and comparing the hit sequence with the seed sequence at positions of the seed sequence that correspond to at least 5 primary complementary surface (PCS) amino acids, wherein each of the at least 5 PCS
amino acids has a hydrogen bond interaction or a van der Waals interaction with ligand when ligand is bound to the first protein; and (iv) identifying the second protein to be a ligand-binding protein if the hit sequence comprises at least 5 amino acids that are consistent with the PCS.
In some embodiments, the spectral shift comprises a monochromatic fluorescence intensity change or a dichromatic spectral shift.
Also provided is a method of converting a biosensor that shows a monochromatic response upon ligand binding into a biosensor with a dichromatic response upon ligand binding, the method comprising (a) selecting a biosensor that exhibits a monochromatic response upon ligand binding, wherein the biosensor comprises a ligand-binding protein and a first reporter group; and (b) attaching a second reporter group to the biosensor, wherein the second reporter group has (i) an excitation spectrum that overlaps with the emission spectrum of the first reporter group; or (ii) an emission spectrum that overlaps with the excitation spectrum of the first reporter group.
The present subject matter also includes method of converting a biosensor that shows a monochromatic response upon ligand binding into a biosensor with a dichromatic response upon ligand binding, the method comprising (a) selecting a biosensor that exhibits a monochromatic response upon ligand binding, wherein the biosensor comprises a ligand-binding fluorescent protein; and (b) attaching an acceptor fluorophore or a donor fluorophore to the biosensor, wherein (i) the acceptor fluorophore has an excitation spectrum that overlaps with the emission spectrum of the fluorescent protein; or (ii) the donor fluorophore has an emission spectrum that overlaps with the excitation spectrum of the fluorescent protein.
Also provided is a method of increasing a dichromatic response of a biosensor to ligand binding, the method comprising (a) selecting a biosensor that exhibits a dichromatic response upon ligand binding, wherein the biosensor comprises a ligand-binding protein and a first reporter group; and (b) attaching a second reporter group to the biosensor, wherein the second reporter group has (i) an excitation spectrum that overlaps with the emission spectrum of the first reporter group; or (ii) an emission spectrum that overlaps with the excitation spectrum of the first reporter group.
In some embodiments, the second reporter group is within about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 4, 6, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, or 200 angstroms (A) of the first reporter group regardless of whether ligand is bound to the biosensor. Suitable distances may be determined in part by the distance-dependence of the energy transfer between a given donor-acceptor pair (see, e.g, J.R. Lakowicz, 2006, Principles of Fluorescence Spectroscopy, Springer, incorporated herein by reference). In some embodiments, when the ligand is bound to the biosensor, the average distance between the first reporter group and the second reporter group changes by less than about 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, or 0.01 angstroms (A) compared to when ligand is not bound to the ligand-binding protein.
The present subject matter further provides a method of converting a biosensor that shows a monochromatic response upon ligand binding into a biosensor with a dichromatic response upon ligand binding, the method comprising (a) selecting a biosensor that exhibits a monochromatic response upon ligand binding, wherein said biosensor comprises an amino acid or polypeptide and a first reporter group, wherein the first reporter group comprises a chemoresponsive fiuorophore; and (b) attaching a second reporter group to said biosensor, wherein said second reporter group has (i) an excitation spectrum that overlaps with the emission spectrum of said first reporter group; or (ii) an emission spectrum that overlaps with the excitation spectrum of said first reporter group.
Also included is a method of increasing a dichromatic response of a biosensor to ligand binding, the method comprising (a) selecting a biosensor that exhibits a dichromatic response upon ligand binding, wherein said biosensor comprises an amino acid or a polypeptide and a first reporter group, wherein the first reporter group comprises a chemoresponsive fiuorophore; and (b) attaching a second reporter group to said biosensor, wherein said second reporter group has (i) an excitation spectrum that overlaps with the emission spectrum of said first reporter group; or (ii) an emission spectrum that overlaps with the excitation spectrum of said first reporter group.

tgmFRET Biosensors While ngmFRET is preferred to tgmFRET, tgmFRET may be used alternatively or in addition to ngmFRET in certain embodiments.
In various embodiments, the biosensor comprises multiple reporter groups, including a first reporter group and a second reporter group. For example, the first reporter group may comprise a donor fluorophore and the second reporter group may comprise an acceptor fluorophore. In certain embodiments, FRET is detectable by a change in the fluorescence of the acceptor fluorophore or by a decrease in donor fluorophore fluorescence.
In various embodiments, the donor fluorophore, and/or the acceptor fluorophore is fluorescent. In some embodiments, both the donor fluorophore and the acceptor fluorophore are fluorescent.
In various embodiments, the angle and/or distance between the donor fluorophore and the acceptor fluorophore changes upon ligand binding. In some embodiments, neither the donor fluorophore nor the acceptor fluorophore is directly responsive to ligand binding. In some embodiments the donor fluorophore and/or the acceptor fluorophore is attached to the N-terminus or the C-terminus of the ligand-binding protein (e.g., directly or via a fluorophore attachment motif). In certain embodiments, the donor fluorophore and/or the acceptor fluorophore is attached to a fluorophore attachment motif. For example, the fluorophore attachment motif may be conjugated to the N-terminus or the C-terminus of the ligand-binding protein.
In some embodiments, the donor fluorophore and/or the acceptor fluorophore comprises a fluorescent protein. In various embodiments, the donor fluorophore and/or the acceptor fluorophore comprises an organic compound having a molecular weight less than about 2000 Da (e.g., 5-iodoacetamidofluorescein (5-IAF) or 6-iodoacetamidofluorescein (6-IAF), rhodamine, Oregon Green, eosin, Texas Red, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, merocyanine, Badan, Acrylodan, IAEDANS, comprising 3-cyano-7-hydroxycoumarin, 7-hydroxycoumarin-3-carboxylic acid, 6,8-difluoro-7-hydroxy-methylcoumarin, or 7-amino-4-methylcoumarin, pyridyloxazole, nitrobenzoxadiazole, benzoxadiazole, DRAQ5, DRAQ7, or CyTRAK Orange, cascade blue, Nile red, Nile blue, cresyl violet, oxazine 170, proflavin, acridine orange, acridine yellow, auramine, crystal violet, malachite green, porphin, phthalocyanine, bilirubin, pyrene, N,N-dimethyl-N-(iodoacety1)-N'-(7-nitrobenz-2-ox- a-1,3-diazol-4-ypethylenediamide (NBD), N-((2-(iodoacetoxy)ethyl)-N-methy- 1)amino-7-nitrobenz-2-oxa-1,3-diazole (NBDE), Acrylodan, JPW4039, JPW4042, JPW4045, Oregon Green, Pacific Blue, CPM, N,N'-Dimethyl-N-(Iodoacety1)-N'-(7-Nitrobenz-2-Oxa-1,3-Diazol-4-y1)Ethylenediamine (IANBD), 7-diethylamino-3-(4'-maleimidylpheny1)-4-methylcoumarin (CPM), BODIPY 499, BODIPY
507/545, BODIPY 499/508, Alexa 432, A1exa488, A1exa532, A1exa546, Cy5, or 1-(2-maleimidylethyl)-4-(5-(4-methoxyphenypoxazol-2- yppyridinium methanesulfonate (PyMPO maleimide) (PyMPO)). For example, the organic compound is a fluorophore.
Numerous combinations of donor and acceptor fluorophores are possible.
Reporter Group Attachment Aspects of the present subject matter provide a biosensor that comprises a one or more reporter groups attached to a ligand-binding protein, wherein binding of a ligand to a ligand-binding domain of the ligand-binding protein causes a change in signaling by the reporter group. In various embodiments, the reporter group is attached to an endosteric site, an allosteric site, or a peristeric site of the ligand-binding protein. In embodiments, the reporter group is covalently or noncovalently attached to the ligand-binding protein.
For convenience and depending on context, a reporter group may be referred to by a name of an unattached form of the reporter group regardless of whether the reporter group is attached to a ligand-binding protein. For example, a compound known as "Compound A"
when in an unconjugated form may be referred to herein as "Compound A" when in a form that is attached to a ligand-binding protein. In a specific example, the term "Acrylodan" is used to refer to unreacted/unconjugated Acrylodan, as well as Acrylodan that is conjugated to a ligand-binding protein.
In certain embodiments, a biosensor comprises a reporter group that is conjugated to a ligand-binding protein, and the reporter group is conjugated to an amino acid of the protein that is at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 angstroms (A) from the ligand when the ligand is bound to the protein. In embodiments, the reporter group is conjugated to an amino acid of the protein that is about 0.1 A to about 5 A, about 5 A to about 10 A, about 10 A to about 20 A, about 20 A to about 50 A, about 50 A to about 75 A, or about 75 A to about 100 A from the ligand when the ligand is bound to the protein. In some embodiments, the reporter group is conjugated to an amino acid of the protein that is within an a-helix or a J3-strand. In some embodiments, the reporter group is conjugated to an amino acid that (i) is not within an a-helix or a J3-strand, but is within about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids of an amino acid of the protein's amino acid sequence that is within an a-helix or a J3-strand. In some embodiments, the reporter group is conjugated to an amino acid that is in an inter-domain hinge amino acid region between two domains of a protein. In some embodiments, the reporter group is conjugated to an amino acid that is in an inter-domain hinge amino acid region between (i) a a-helix and a J3-strand; (ii) two a-helixes; or (iii) two J3-strands of a protein. In some embodiments, the reporter group is conjugated to an amino acid (e.g., a cysteine such as a cysteine added by substitution compared to a naturally corresponding polypeptide) between positions 1-25, 25-50, 50-75, 75-100, 100-125, 125-150, 150-175, 175-200, 200-225, 225-250, 250-275, 275-350, 275-300, 275-325, 300-325, 300-350, 300-400, or 350-450 (inclusive) of a polypeptide (e.g., not including N-terminal fusion proteins compared to the polypeptide's naturally occurring counterpart).
In certain embodiments, the directly or indirectly responsive fluorophore is conjugated (directly or via a fluorophore attachment motif) to an amino acid that is no more than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 5-15, 5-20, 5-25, 5-100, 10-15, 10-20, 10-25, 10-50, 10-100, 25-50, 25-75, or 25-100 amino acids from the N-terminus or the C-terminus of the ligand-binding protein. In some embodiments, the directly or indirectly responsive fluorophore is conjugated (directly or via a fluorophore attachment motif) to an amino acid that is at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 5-15, 5-20, 5-25, 5-100, 10-

15, 10-20, 10-25, 10-50, 10-100, 25-50, 25-75, or 25-100 amino acids from the N-terminus or the C-terminus of the ligand-binding protein. In some embodiments, about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 5-15, 5-20, 5-25, 5-100, 10-15, 10-20, 10-25, 10-50, 10-100, 25-50, 25-75, or 25-100 amino acids (including or not including the signal peptide) have been deleted (e.g. are absent) from the N-terminus of the protein compared to its naturally occurring counterpart. In some embodiments, less than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 5-15, 5-20, 5-25, 5-100, 10-15, 10-20, 10-25, 10-50, 10-100, 25-50, 25-75, or 25-100 amino acids (including or not including the signal peptide) have been deleted (e.g. are absent) from the N-terminus of the protein compared to its naturally occurring counterpart. In some embodiments, about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 5-15, 5-20, 5-25, 5-100, 10-15, 10-20, 10-25, 10-50, 10-100, 25-50, 25-75, or 25-100 amino acids have been deleted (e.g. are absent) from the C-terminus of the protein compared to its naturally occurring counterpart. In some embodiments, less than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 5-15, 5-20, 5-25, 5-100, 10-15, 10-20, 10-25, 10-50, 10-100, 25-50, 25-75, or 25-100 amino acids have been deleted (e.g. are absent) from the C-terminus of the protein compared to its naturally occurring counterpart.
Periplasmic binding proteins are characterized by two lobes connected by a hinge region; ligand bind at a location at the interface between the two domains.
Such proteins or engineered versions thereof (as described herein) can adopt two different conformations: a ligand-free open form and a ligand-bound closed form, which interconvert through a relatively large bending motion around the hinge (FIG. 1A; Dwyer et al., 2004, Current Opinion in Structural Biology 12:495-504).
The remarkable adaptability of this superfamily of ligand-binding proteins is likely to have arisen from positioning the location of binding of the ligand at the interface between the lobes and from the large ligand-mediated conformational change. In this arrangement, ligands are placed within an environment that resembles a protein interior, but the residues forming the contact points or contact sites with the ligand are positioned at the surface of the lobes.
Direct signaling relationships between proteins and reporter groups are readily designed by replacing a residue known to form a ligand contact with a cysteine to which the fluorophore is attached ("endosteric" attachment site). Other, indirect signaling relationships can be established in two ways. The first relies on visual inspection of the ligand complex structure, and identifying residues that are located in the vicinity of the binding site, but do not interact directly with the ligand, and that are likely to be involved in conformational changes. Typically, such "peristeric" sites are located adjacent to the residues that form direct contacts with the bound ligand. In the case of the bPBPs, such residues are located at the perimeter of the inter-domain cleft that forms the ligand binding site location. The environment of these peristeric sites changes significantly upon formation of the closed state.
These are examples of positions which are proximal to the ligand-binding pocket/domain.
The second, most general, approach identifies sites in the protein structure that are located anywhere in the protein, including locations at some distance away from the ligand-binding site (i.e., distal to the ligand-binding pocket/domain), and undergo a local conformational change in concert with ligand binding. If the structures of both the open and closed states are known, then such "allosteric" sites can be identified using a computational method that analyzes the conformational changes that accompany ligand binding (Marvin et al., Proc.
Natl. Acad. Sci. USA 94:4366-4371, 1997). Alternatively, once allosteric sites have been identified in one bPBP, modeling and structural homology arguments can be invoked to identify such sites in other bPBPs in which only one state has been characterized (Marvin &
Hellinga, J. Am. Chem. Soc. 120:7-11, 1998). This generalized conformational analysis also may identify peristeric and endosteric sites, which were identified and classified by visual inspection. The domain or region involved in ligand binding is comprised of a plurality of residues, e.g., non-contiguous amino acids of the ligand-binding protein, which are contact points or sites of contact between the ligand and its cognate ligand-binding protein.
In non-limiting implementations, the reporter group is attached to the ligand-binding protein via a biotin-avidin interaction. The reporter group may be, e.g., conjugated to biotin and the ligand-binding protein is conjugated to avidin. In an example, the avidin is bound to four biotin molecules wherein each biotin molecule is individually conjugated to a reporter group. Alternatively, the reporter group is conjugated to avidin and the ligand-binding protein is conjugated to biotin. For example, the avidin is bound to four biotin molecules, wherein each biotin molecule is individually conjugated to a ligand-binding protein.
As used herein, "conjugated" means covalently attached. One compound may be directly conjugated to another compound, or indirectly conjugated, e.g., via a linker.
In some embodiments, the reporter group is directly attached to the ligand-binding protein. In various embodiments, the reporter group is attached to an amino acid of the ligand-binding protein that is at least about 2, 4, 6, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 angstroms (A) from the ligand when the ligand is bound to the ligand-binding protein. In certain embodiments, the reporter group is conjugated to an amino acid having a position within positions 1-25, 25-50, 50-75, 75-100, 100-125, 125-150, 150-175, 175-200, 200-225, 225-250, 250-275, or 275-300 of the ligand-binding protein, wherein position 1 is the N-terminal amino acid of the ligand-binding protein. In non-limiting examples, the reporter group is conjugated to an amino acid of the ligand-binding protein that is (a) within an a-helix or a J3-strand of the ligand-binding protein; (b) not within an a-helix; (c) not within a J3-strand; (d) within about 5 or 10 amino acids of an amino acid that is within an a-helix or J3-strand; (e) within a stretch of consecutive amino acids that links two domains of the ligand-binding protein; (f) within a stretch of consecutive amino acids that links an a-helix and a J3-strand; (g) within a stretch of consecutive amino acids that links two a-helices; or (h) within a stretch of consecutive amino acids that links two J3-strands. In some embodiments, the reporter group is directly attached to the N-terminus or the C-terminus of the ligand-binding protein.
The reporter group may be conjugated to the ligand-binding protein a variety of linkers or bonds, including (but not limited to) a disulfide bond, an ester bond, a thioester bond, an amide bond, or a bond that has been formed by a click reaction. In some embodiments, the click reaction is a reaction between (a) an azide and an alkyne; (b) an azide and an alkyne in the presence of Cu(I); (c) an azide and a strained cyclooctyne; (d) an azide and a dibenzylcyclooctyne, a difiuorooctyne, or a biarylazacyclooctynone; (e) a diaryl-strained-cyclooctyne and a 1,3-nitrone; (f) an azide, a tetrazine, or a tetrazole and a strained alkene; (g) an azide, a tetrazine, or a tretrazole and a oxanorbomadiene, a cyclooctene, or a trans-cycloalkene; (h) a tetrazole and an alkene; or (i) a tetrazole with an amino or styryl group that is activated by ultraviolet light and an alkene. These exemplary click chemistry reactions have high specificity, efficient kinetics, and occur in vivo under physiological conditions. See, e.g., Baskin et al. Proc. Natl. Acad. Sci. USA
104(2007):16793; Oneto et al.
Acta biomaterilia (2014); Neves et al. Bioconjugate chemistry 24(2013):934;
Koo et al.
Angewandte Chemie 51(2012):11836; Rossin et al. Angewandte Chemie 49(2010):3375, and U.S. Patent Application Publication No. 20160220686, published August 4, 2016, the entire content of each of which is incorporated herein by reference. For a review of a wide variety of click chemistry reactions and their methodologies, see e.g., Nwe K and Brechbiel M W, 2009 Cancer Biotherapy and Radiopharmaceuticals, 24(3): 289-302; Kolb H C et al., 2001 Angew. Chem. Int. Ed. 40: 2004-2021. The entire contents of each of the foregoing references are incorporated herein by reference.
As used herein, the term "linker" refers to a molecule or sequence (such as an amino acid sequence), that attaches, as in a bridge, one molecule or sequence to another molecule or sequence. "Linked" means attached or bound by covalent bonds, or non-covalent bonds, or other bonds, such as van der Waals forces. In some embodiments, a linker comprises a chemical structure that has resulted from a reaction used to attach one molecule to another.
In various implementations of the present subject matter, the reporter group is conjugated to a cysteine of the ligand-binding protein. The cysteine may be present on a natural counterpart or version of the ligand-binding protein or added to the ligand-binding protein by a substitution mutation. In some embodiments, the cysteine is at the N-terminus or the C-terminus of the ligand-binding protein.

Non-limiting examples relate to the conjugation of a reporter group to a primary amine of the ligand-binding protein. In certain embodiments, the primary amine is present in a lysine of the ligand-binding protein. The lysine may be present on a natural counterpart or version of the ligand-binding protein or added to the ligand-binding protein by a substitution mutation. In various embodiments, the lysine is at the N-terminus or the C-terminus of the ligand-binding protein.
Aspects of the present subject matter provide a biosensor in which the reporter group is attached to the ligand-binding protein via a linker. In some embodiments, the linker comprises an organic compound that is less than about 30, 20, 15, or 10 A
long. Non-limiting examples of linkers include 0, S, NH, PH, and alkyl linkers.
"Alkyl," as used herein, refers to the radical of saturated or unsaturated aliphatic groups, including straight-chain alkyl, alkenyl, or alkynyl groups, branched-chain alkyl, alkenyl, or alkynyl groups, cycloalkyl, cycloalkenyl, or cycloalkynyl (alicyclic) groups, alkyl substituted cycloalkyl, cycloalkenyl, or cycloalkynyl groups, and cycloalkyl substituted alkyl, alkenyl, or alkynyl groups. Unless otherwise indicated, a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., C1-C30 for straight chain, C3-C30 for branched chain), more preferably 20 or fewer carbon atoms, more preferably 12 or fewer carbon atoms, and most preferably 8 or fewer carbon atoms. Likewise, preferred cycloalkyls have from 3-10 carbon atoms in their ring structure, and more preferably have 5, 6 or 7 carbons in the ring structure. The ranges provided above are inclusive of all values between the minimum value and the maximum value. The term "alkyl" includes both "unsubstituted alkyls" and "substituted alkyls," the latter of which refers to alkyl moieties having one or more substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone.
Such substituents include, but are not limited to, halogen, hydroxyl, carbonyl (such as a carboxyl, alkoxycarbonyl, formyl, or an acyl), thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, a phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, or an aromatic or heteroaromatic moiety. Unless the number of carbons is otherwise specified, "lower alkyl" as used herein means an alkyl group, as defined above, but having from one to ten carbons, more preferably from one to six carbon atoms in its backbone structure. Likewise, "lower alkenyl" and "lower alkynyl" have similar chain lengths. Preferred alkyl groups are lower alkyls.
The alkyl groups may also contain one or more heteroatoms within the carbon backbone.
Preferably the heteroatoms incorporated into the carbon backbone are oxygen, nitrogen, sulfur, and combinations thereof. In certain embodiments, the alkyl group contains between one and four heteroatoms.
In some embodiments, the linker comprises a bond formed by a chemical reaction involving a reactive group such as a maleimide group. Alternatively or in addition, the linker comprises a stretch of amino acids. In a non-limiting example, the linker comprises a polyglycine linker. In embodiments, the polyglycine linker comprises 2, 3, 4, 5, or more glycines. Optionally, the polyglycine linker further comprises a serine.
In various implementations, the reporter group is attached to a linker via a covalent bond and the linker is attached to a ligand-binding protein via a covalent bond. In embodiments, the covalent bond between the linker and the reporter group and/or the covalent bond between the linker and the ligand-binding protein is a disulfide bond, an ester bond, a thioester bond, an amide bond, a carbamate bond, or a bond that has been formed by a click reaction. Non-limiting examples of click reactions include reactions between an azide and an alkyne; an azide and an alkyne in the presence of Cu(I); an azide and a strained cyclooctyne; an azide and a dibenzylcyclooctyne, a difluorooctyne, or a biarylazacyclooctynone; a diaryl-strained-cyclooctyne and a 1,3-nitrone; an azide, a tetrazine, or a tetrazole and a strained alkene; an azide, a tetrazine, or a tretrazole and a oxanorbomadiene, a cyclooctene, or a trans-cycloalkene; a tetrazole and an alkene; or a tetrazole with an amino or styryl group that is activated by ultraviolet light and an alkene.
The present subject matter also includes biosensors having one or more reporter groups attached to a ligand-binding protein via a fluorophore attachment motif.
Fluorophore Attachment Motifs Aspects of the present subject matter include the use of one or more fluorophore attachment motifs to attach one or more reporter groups to a ligand-binding protein. For example, a reporter group may be attached to a fluorophore attachment motif that is attached to the N-terminus or the C-terminus of the ligand-binding protein.
In various implementations, the fluorophore attachment motif comprises a polypeptide. In some embodiments, the polypeptide comprises amino acids in the PZif amino acid sequence (SEQ ID NO: 42).
In some embodiments, the polypeptide comprises a stretch of at least 50, 60, 70, 80, 90, or 100 amino acids in a sequence that is at least about 85%, 90%, 95%, or 99% identical to the amino acid sequence of E. coli thioredoxin (ecTRX; SEQ ID NO: 151). In some embodiments, the polypeptide is a mutant of ecTRX comprising a D3X, K4X, K1 9X, D27X, K37X, K53X, K58X, K70X, R74X, K83X, K91X, K97X, or K101X mutation, or any combination thereof, wherein X is any amino acid, and wherein each ecTRX amino acid position is numbered as in SEQ ID NO: 151. In certain embodiments, the polypeptide is a mutant of ecTRX comprising a D3A, K4R, K4Q, K19R, K19Q, D27A, K37R, K53M, K53R, K58M, K7OR, R74C, K83R, K91R, K97R, or K101R mutation, or any combination thereof, wherein each ecTRX amino acid position is numbered as in SEQ ID NO: 151.
In non-limiting examples, the polypeptide comprises amino acids in the sequence of any one of SEQ ID NOS: 69-86 or 151.
In certain embodiments, the polypeptide comprises (a) at least 1, 2, or 3 thiol groups;
(b) at least 1, 2, or 3 cysteines that each comprise a sulfhydryl group; (c) at least 1, 2, or 3 primary amine groups; and/or (d) at least 1, 2, or 3 lysines that each comprise a primary amine. In some embodiments there is no disulfide bond between cysteines within the amino acid sequence of the polypeptide.
In some embodiments, the polypeptide comprises a hexahistidine tag. In some embodiments, the hexahistidine tag is attached to another portion of the polypeptide via a GGS linker.
Reporter Groups Various types of reporter groups may be used in embodiments of the present subject matter. For example, the reporter group may comprise a fluorophore that produces a fluorescent signal. Biosensors comprising a fluorophore may be referred to herein as fluorescently responsive sensors (FRSs).
Preferably, the binding of ligand to an FRS results in a change in ratiometric AR in the signal from a reporter group. A ratiometric signal (R1,2) is defined as the quotient of two intensities, /xi and Ix2, measured at two independent wavelengths, k1 and k2 and may be calculated according to the following equation:
R1,2 = -1,11 /-1,12 The two independent wavelengths Xi and k2 may be from a single fluorophore or from a combination of two or more fluorophores (e.g., a pair of fluorophores between which tgmFRET and/or ngmFRET occurs). In some embodiments, k1 falls within the emission spectrum of a directly responsive fluorophore and k2 falls within the emission spectrum of an indirectly responsive fluorophore. In certain embodiments, Xi falls within the emission spectrum of an indirectly responsive fluorophore and k2 falls within the emission spectrum of a directly responsive fluorophore. In various embodiments, k1 falls within the emission spectrum of both a directly responsive fluorophore and an indirectly responsive fluorophore.
In various embodiments, k2 falls within the emission spectrum of both a directly responsive fluorophore and an indirectly responsive fluorophore.
In some embodiments, intensities are, e.g., integrated, filtered, assessed, detected, or evaluated over a range of wavelengths. In some embodiments, intensities are integrated over a range of wavelengths in a recorded emission spectrum. In some embodiments, a range of wavelengths is selected using a filter. In some embodiments, k1 is the intensity over a 1 nm to 60 nm interval centered between 400 and 1000 nm, and k2 is the intensity over a 1 nm to 60 nm interval centered between 400 nm and 1000 nm. In some embodiments, intensities are integrated, filtered, assessed, detected, or evaluated over a 1 nm, 2 nm, lOnm, 15nm, 20nm, 25nm, 30nm, 35nm, 40nm, 45nm, 50nm, 55 nm, 60 nm, 75 nm, 100 nm, 10-40 nm, 10-nm, 20-50 nm, or 10-100 nm regions, centered between 400-1000 nm, e.g. between 420 nm and 520 nm for k1, and 400-1000nm, e.g. between 500 nm to 600 nm for k2. In some embodiments, intensities are recorded through a bandpass filter. A non-limiting example of a bandpass filter is a 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 75 nm, 100 nm, 10-40 nm, 10-50 nm, 20-50 nm, or 10-100 nm bandpass filter, centered between 400-1000 nm, e.g. at 452 nm forki and at 400-1000nm, e.g. at 528 nm (k2).
Aspects of the present subject matter provide FRSs whose emission spectra change (e.g., the shape of the emission spectra change) in response to ligand binding. In various embodiments, the ratio of intensities at two chosen wavelengths of an FRS's emission spectrum changes upon ligand binding.
In various embodiments, the emission spectra of two or more fluorophores contributes to /xi and/or A2. In some embodiments, the emission spectrum of a directly responsive fluorophore contributes to Ai and/or A2 and the emission spectrum of an indirectly responsive fluorophore contributes to Ai and/or A2. In certain embodiments, a directly responsive fluorophore contributes to Ai and the emission spectrum of an indirectly responsive fluorophore contributes to A2. In some embodiments, a directly responsive fluorophore contributes to A2 and the emission spectrum of an indirectly responsive fluorophore contributes to Ai. In various embodiments, both the emission spectrum of a directly responsive fluorophore and the emission spectrum of an indirectly responsive fluorophore contributes to Ai. In some embodiments, both the emission spectrum of a directly responsive fluorophore and the emission spectrum of an indirectly responsive fluorophore contributes to I.
In some embodiments, the emission wavelength and/or intensity of a fluorophore (e.g., a single fluorophore in a biosensor comprising one reporter group or a directly responsive fluorophore comprising reporter groups between which tgmFRET and/or ngmFRET occurs) changes when the positions of atoms within the fluorophore change with respect to each other (e.g., due to the rotation of bound atoms with respect to each other or a change in the angle of a bond). In non-limiting examples, the emission wavelength and/or intensity of the fluorophore changes when (i) one portion of the fluorophore rotates around a bond axis compared to another portion of the fluorophore and/or (ii) when the angle of a bond between two atoms of the fluorophore changes. In a non-limiting example, the fluorophore is a prodan-derived fluorophore (e.g., Acrylodan or Badan) and binding of ligand alters the orientation of a dimethylamino group, a naphthalene ring, and/or a carbonyl with respect to the ligand-binding protein and/or each other. In a non-limiting example, the degree of polarization of a dipole on the fluorophore changes in response to ligand binding. In various embodiments, the emission wavelength and/or intensity of the fluorophore changes when an atom electrostatically interacts with the fluorophore. For example, the emission wavelength and/or intensity of the fluorophore changes when the source of a positive or negative charge changes its distance with respect to the fluorophore within about 1, 2, 3, 4, 5, or 10 A of the fluorophore. In some embodiments, the fluorophore exhibits hypsochromicity or bathochromicity upon ligand binding to the ligand-binding domain of the ligand-binding protein. In certain embodiments, the fluorophore has an emission spectrum comprising radiation with a wavelength (e.g., a peak emission wavelength) of about 400 nm, 410 nm, 420 nm, 430 nm, 440 nm, 450 nm, 460 nm, 470 nm, 480 nm, 490 nm, 500 nm, 510 nm, 520 nm, 530 nm, 540 nm, 550 nm, 560 nm, 570 nm, 580 nm, 590 nm, 600 nm, 610 nm, 620 nm, 630 nm, 640 nm, 650 nm, 660 nm, 670 nm, 680 nm, 690 nm, 700 nm, 710 nm, 720 nm, 730 nm, 740 nm, 750 nm, 760 nm, 770 nm, 780 nm, 790 nm, 800 nm, 850 nm, 900 nm, 950 nm, or 1000 nm, or about 400 nm to about 450 nm, about 450 nm to about 500 nm, about 500 nm to about 550 nm, about 550 nm to about 600 nm, about 600 nm to about 650nm, about 650 to about 700 nm, about 700 nm to about 750 nm, about 750 nm to about 800 nm, or about 800 nm to about 1000 nm.
In some embodiments, the signal comprises the emission intensity of the FRS
recorded at a single wavelength or range of wavelengths. The change in signal may be a shift in the single wavelength or range of wavelengths. In some embodiments, the shift in the wavelength is at least about 1 nm, at least about 2 nm, at least about 3 nm, at least about 4 nm, at least about 5 nm, at least about 6 nm, at least about 7 nm, at least about 8 nm, at least about 9 nm, at least about 10 nm, at least about 11 nm, at least about 12 nm, at least about 13 nm, at least about 14 nm, at least about 15 nm, at least about 16 nm, at least about 17 nm, at least about 18 nm, at least about 19 nm, at least about 20 nm, at least about 25 nm, at least about 30 nm, at least about 35 nm, at least about 40 nm, at least about 45 nm, at least about 50 nm, at least about 55 nm, at least about 60 nm, at least about 65 nm, at least about 70 nm, at least about 75 nm, at least about 80 nm, at least about 85 nm, at least about 90 nm, at least about 95 nm, at least about 100 nm, at least about 105 nm, at least about 110 nm, at least about 115 nm, at least about 120 nm, at least about 125 nm, or at least about 130 nm. In some embodiments, the shift in the wavelength is about 1 nm to about 20 nm, about 2 nm to about nm, about 3 nm to about 20 nm, about 4 nm to about 20 nm, about 5 nm to about 20 nm, about 1 nm to about 19 nm, about 1 nm to about 18 nm, about 1 nm to about 17 nm, 1 nm to about 16 nm, about 1 nm to about 15 nm, about 1 nm to about 14 nm, about 1 nm to about 13 20 nm, about 1 nm to about 12 nm, about 1 nm to about 11 nm, or about 1 nm to about 10 nm. In some embodiments, the shift in the wavelength is about 1 nm to about 20 nm. In some embodiments, the shift in the wavelength is about 1 nm to about 130 nm.
In certain embodiments, the signal comprises the ratio or quotient of the emission intensities recorded at two distinct wavelengths or ranges of wavelengths, i.e. , a ratiometric signal. For example, as shown in FIG. 1, ligand binding may be determined by measuring the ratio of blue to green emission intensities. The change in signal may be decreased emission intensity at one wavelength, and no change in emission intensity at the other wavelength. The change in signal may be increased emission intensity at one wavelength, and no change in emission intensity at the other wavelength. The change in signal may be increased emission intensity at one wavelength, and increased emission intensity at the other wavelength. The change in signal may be decreased emission intensity at one wavelength, and decreased emission intensity at the other wavelength. The change in signal may be increased emission intensity at one wavelength, and decreased emission intensity at the other wavelength. In some embodiments, the change in ratio of the emission intensities recorded at two distinct wavelengths or ranges of wavelengths may be at least about 1.1-fold, at least about 1.2-fold, at least about 1.4-fold, at least about 1.6-fold, at least about 1.8-fold, at least about 2.0-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 5.5-fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8-fold, at least about 8.5-fold, at least about 9-fold, at least about 9.5-fold, at least about 10-fold, at least about 12-fold, at least about 14-fold, at least about 16-fold, at least about 18-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, at least about 50-fold, at least about 55-fold, at least about 60-fold, at least about 65-fold, at least about 70-fold, at least about 75-fold, at least about 80-fold, at least about 85-fold, at least about 90-fold, at least about 95-fold, or at least about 100-fold. In some embodiments, the change in ratio of the emission intensities recorded at two distinct wavelengths or ranges of wavelengths may be a decrease of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, or of 5-25%, 25-50%, 25-75%, 50-75%, 50-90%, or 75-99%
or the reciprocal thereof.
The change in signal may be a change in the ratio of the two distinct wavelengths or ranges of wavelengths. The change in signal may be a shift in the two distinct wavelengths or ranges of wavelengths. In some embodiments, one wavelength shifts. In some embodiments, both wavelengths shift. In some embodiments, the shift in the wavelength is at least about 1 nm, at least about 2 nm, at least about 3 nm, at least about 4 nm, at least about 5 nm, at least about 6 nm, at least about 7 nm, at least about 8 nm, at least about 9 nm, at least about 10 nm, at least about 11 nm, at least about 12 nm, at least about 13 nm, at least about 14 nm, at least about 15 nm, at least about 16 nm, at least about 17 nm, at least about 18 nm, at least about 19 nm, at least about 20 nm, at least about 25 nm, at least about 30 nm, at least about 35 nm, at least about 40 nm, at least about 45 nm, at least about 50 nm, at least about 55 nm, at least about 60 nm, at least about 65 nm, at least about 70 nm, at least about 75 nm, at least about 80 nm, at least about 85 nm, at least about 90 nm, at least about 95 nm, at least about 100 nm, at least about 105 nm, at least about 110 nm, at least about 115 nm, at least about 120 nm, at least about 125 nm, or at least about 130 nm. In some embodiments, the shift in the wavelength is about 1 nm to about 20 nm, about 2 nm to about 20 nm, about 3 nm to about 20 nm, about 4 nm to about 20 nm, about 5 nm to about 20 nm, about 1 nm to about 19 nm, about 1 nm to about 18 nm, about 1 nm to about 17 nm, 1 nm to about 16 nm, about 1 nm to about 15 nm, about 1 nm to about 14 nm, about 1 nm to about 13 nm, about 1 nm to about 12 nm, about 1 nm to about 11 nm, or about 1 nm to about 10 nm. In some embodiments, the shift in the wavelength is about 1 nm to about 20 nm. In some embodiments, the shift in the wavelength is about 1 nm to about 130 nm.
A fluorophore may comprise, e.g., a fluorescent protein or an organic compound having a molecular weight less than about 2000 Daltons (Da). Non-limiting examples of commercially available fluorophores include such as 5-iodoacetamidofluorescein (5-IAF) or 6-iodoacetamidofluorescein (6-IAF), rhodamine, Oregon Green, eosin, Texas Red, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, merocyanine, Badan, Acrylodan, IAEDANS, comprising 3-cyano-7-hydroxycoumarin, 7-hydroxycoumarin-3-carboxylic acid, 6,8-difluoro-7-hydroxy- 4-methylcoumarin, or 7-amino-4-methylcoumarin, ppidyloxazole, nitrobenzoxadiazole, benzoxadiazole, DRAQ5, DRAQ7, or CyTRAK Orange, cascade blue, Nile red, Nile blue, cresyl violet, oxazine 170, proflavin, acridine orange, acridine yellow, auramine, crystal violet, malachite green, porphin, phthalocyanine, bilirubin, pyrene, N,1\11-dimethyl-N-(iodoacety1)-N'-(7-nitrobenz-2-ox- a-1,3-diazol-4-ypethylenediamide (NBD), N-((2-(iodoacetoxy)ethyl)-N-methy- 1)amino-7-nitrobenz-2-oxa-1,3-diazole (NBDE), Acrylodan, JPW4039, JPW4042, JPW4045, Oregon Green, Pacific Blue, CPM, N,N-Dimethyl-N-(Iodoacety1)-N-(7-Nitrobenz-2-Oxa-1,3-Diazol-4-y1)Ethylenediamine (IANBD), 7-diethylamino-3-(4'-maleimidylpheny1)-4-methylcoumarin (CPM), BODIPY 499, 507/545, BOD1PY 499/508, Alexa 432, A1exa488, A1exa532, A1exa546, Cy5, or 1-(2-maleimidylethyl)-4-(5-(4-methoxyphenypoxazol-2- yppyridinium methanesulfonate (PyMPO maleimide) (PyMPO). In various embodiments, the reporter group was thiol-reactive prior to being conjugated to a polypeptide disclosed herein. In embodiments, the reporter group is linked to a polypeptide disclosed herein via a disulfide bond. Additional non-limiting examples of commercially available fluorophores include fluorescent proteins such as Blue Fluorescent Protein (BFP), TagBFP, mTagBFP2, Azurite, Enhanced Blue Florescent Protein 2 (EBFP2), mKalamal, Sirius, Sapphire, T-Sapphire, Cyan Fluorescent Protein (CFP); Enhanced Cyan Fluorescent Protein (ECFP), Cerulean, SCFP3A, mTurquoise, mTurquoise2, monomeric Midoriishi-Cyan, TagCFP, mTFP1, AmCyanl, Green Fluorescent Protein (GFP), Enhanced Green Fluorescent Protein (EGFP), Emerald, Superfolder GFP, AcGFP1, ZsGreenl, Monomeric Azami Green, TagGFP2, mUKG, mWasabi, Clover, mNeonGreen, Yellow Fluorescent Protein (YFP), Enhanced Yellow Fluorescent Protein (EYFP), Citrine, Venus, Super Yellow Fluorescent Protein 2 (SYFP2), TagYFP, ZsYellowl, mBanana, Orange Fluorescent Protein (OFP), Monomeric Kusabira-Orange (mK0), mKOK, mK02, mOrange, mOrange2, Red Fluorescent Protein (RFP), DsRed-Express, DsRed-Express2, DsRed2, AsRed2, mRaspbeny, mCheny, mStrawberry, mTangerine, tdTomato, TagRFP, TagRFP-T, mApple, mRuby, mRuby2, mPlum, HcRed-Tandem, mKate2, mNeptune, HcRedl, E2-Crimson, NirFP, TagRF'P657, 1FP1.4, or iRFP.
In some embodiments, the fluorophore comprises xanthene, a xanthene derivative, fluorescein, a fluorescein derivative, coumarin, a coumarin derivative, cyanine, a cyanine derivative, rhodamine, a rhodamine derivative, phenoxazine, a phenoxazine derivative, squaraine, a squaraine derivative, coumarin, a coumarin derivative, oxadiazole, an oxadiazole derivative, anthracene, an anthracene derivative, a boradiazaindacine (BODIPY) family fluorophore, pyrene, a pyrene derivative, acridine, an acridine derivative, arylmethine, an arylmethine derivative, tetrapynole, or a tetrapynole derivative. Non-limiting aspects of fluorophores are discussed in Lavis and Raines (2014) ACS Chem. Biol. 9, 855-866, the entire content of which is incorporated herein by reference. For example, the fluorophore may comprise a xanthene derivative comprising fluorescein or a fluorescein derivative, rhodamine, Oregon Green, eosin, or Texas Red. Non-limiting examples of fluorescein derivatives include 5-fluorescein, 6-carboxyfluorescein, 3'6-carboxyfluorescein, 5(6)-carboxyfluorescein, 6-hexachlorofluorescein, 6-tetrachlorofluorescein, or isothiocyanate. In some embodiments, the fluorophore comprises a cyanine derivative comprising indocarbocyanine, oxacarbocyanine, thiacarbocyanine, or merocyanine. In certain embodiments, the fluorophore comprises a squaraine derivative comprising a ring-substituted squaraine. In various embodiments, the fluorophore comprises a naphthalene derivative comprising a dansyl or prodan naphthalene derivative. In a non-limiting example, the fluorophore comprises prodan or a derivative thereof. In certain embodiments, the fluorophore comprises Badan, Acrylodan, or N-(Iodoacetaminoethyl)-1-naphthylamine-5-sulfonic acid (IAEDANS). In some embodiments, the fluorophore comprises a coumarin derivative such as 3-cyano-7-hydroxycoumarin, 7-hydroxycoumarin-3-carboxylic acid, 6,8-difluoro-7-hydroxy- 4-methylcoumarin (DiFMU), or 7-amino-4-methylcoumarin. In various embodiments, the fluorophore comprises an oxadiazole derivative such as pyridyloxazole, nitrobenzoxadiazole, or benzoxadiazole. In certain embodiments, the fluorophore comprises an anthracene derivative comprising an anthraquinone such as DRAQ5, DRAQ7, or CyTRAK Orange. In various embodiments, the fluorophore comprises a pyrene derivative comprising cascade blue. In non-limiting examples the fluorophore comprises an oxazine derivative such as Nile red, Nile blue, cresyl violet, or oxazine 170. In some embodiments, the fluorophore comprises an acridine derivative such as proflavin, acridine orange, or acridine yellow. In certain embodiments, the fluorophore comprises an arylmethine derivative such as auramine, crystal violet, or malachite green. In various embodiments, the fluorophore comprises a tetrapynole derivative comprising porphin, phthalocyanine, or bilirubin.
Aspects of the present subject matter relate to the use of fluorophores that may readily be attached to a ligand-binding protein disclosed herein, e.g., at a cysteine residue. For example, a fluorophore may comprise a sulfhydryl group prior to attachment to a ligand-binding protein that is reacted with a moiety of the ligand-binding protein to attach the fluorophore to the ligand-binding protein. In some embodiments, the fluorophore comprised a thiol group prior to attachment to the ligand-binding protein. For example, the fluorophore was thiol reactive prior to attachment to the ligand-binding protein. Non-limiting examples of fluorophores that may readily be attached to ligand-binding proteins using thiol reactions include fluorescein, pyrene, NBD, NBDE, Acrylodan (6-acryloyl 1-2-dimethylaminonaphthalene), Badan (6-bromo-acetyl-2-dimethylamino-naphthalene), JPW4039, JPW4042, or JPW4045.
In certain embodiments, the fluorophore comprises a derivative of a Prodan-based fluorophore such as Acrylodan or Badan. The excitation and emission properties of the Prodan-based fluorophores Acrylodan and Badan can be altered by manipulating the fluorescent ring system, while preserving the dimethylamino donor group, and the twistable carbonyl acceptor (Klymchenko 2013 Progress in Molecular Biology and Translational Science, 35-58). Replacement of the two-ring naphthalene with a three-ring anthracene (Lu 2006 J. Org. Chem., 71, 9651-9657), fluorene (Kucherak 2010 J. Phys. Chem.
Lett., 1, 616-620), pyrene (Niko 2013 Chem. Eur. J, 19, 9760-9765), or styrene (Benedetti 2012 J. Am.
Chem. Soc., 134, 12418-12421) cores significantly red-shift the excitation and emission properties, and in the case of the latter two, improve brightness through improvements in their excitation peak extinction coefficients. The entire content of each of the references cited above (as well as all other references referred to herein including the contents of nucleic acid and amino acid sequence accession number references) are incorporated herein by reference. Non-limiting examples of prodan analogues include 2-cyano-6-dihexylaminoanthracene and 2-propiony1-6-dihexylaminoanthracene, as well as fluorophores comprising the following structures:
j N1\1' 4,11 fi \
Itr Q.

or In some embodiments, the fluorophore comprises a fluorescent protein.
Fluorescent proteins that emit blue, cyan, green, yellow, orange, red, far-red, or near infrared radiation when contacted with excitation radiation are known in the art and commercially available as proteins and via the expression of vectors that encode the fluorescent protein. Non-limiting examples of fluorescent proteins include Blue Fluorescent Protein (BFP), TagBFP, mTagBFP2, Azurite, Enhanced Blue Florescent Protein 2 (EBFP2), mKalamal, Sirius, Sapphire, T-Sapphire, Cyan Fluorescent Protein (CFP); Enhanced Cyan Fluorescent Protein (ECFP), Cerulean, SCFP3A, mTurquoise, mTurquoise2, monomeric Midoriishi-Cyan, TagCFP, mTFP1, AmCyanl, Green Fluorescent Protein (GFP), Enhanced Green Fluorescent Protein (EGFP), Emerald, Superfolder GFP, AcGFP1, ZsGreenl, Monomeric Azami Green, TagGFP2, mUKG, mWasabi, Clover, mNeonGreen, Yellow Fluorescent Protein (YFP), Enhanced Yellow Fluorescent Protein (EYFP), Citrine, Venus, Super Yellow Fluorescent Protein 2 (SYFP2), TagYFP, ZsYellowl, mBanana, Orange Fluorescetn Protein (OFP), Monomeric Kusabira-Orange (mK0), mKOK, mK02, mOrange, mOrange2, Red Fluorescent Protein (RF'P), DsRed-Express, DsRed-Express2, DsRed2, AsRed2, mRaspberry, mCherry, mStrawbeny, mTangerine, tdTomato, TagRF'P, TagRF'P-T, mApple, mRuby, mRuby2, mPlum, HcRed-Tandem, mKate2, mNeptune, HcRedl, E2-Crimson, NirFP, TagRF'P657, 1FP1.4, or iRFP.
In some embodiments, the fluorophore comprises a quantum dot (Medintz et al.
2005) (Sapsford, Beni and Medintz 2006 Angew Chem Int Ed Engl, 45, 4562-89; Resch-Genger et al. 2008 Nat Methods, 5, 763-75). In some embodiments the emission properties of the conjugated protein are enhanced by immobilization on or near metallic nanoparticles (Zeng et al. 2014 Chem Soc Rev, 43, 3426-52; Shen et al. 2015 Nanoscale, 7, 20132-41).
In various embodiments, the peak emission wavelength and/or the emission intensity of the biosensor change when the ligand binds to the ligand-binding protein.
In some embodiments, the biosensor exhibits a dichromatic signaling change when the ligand binds to the ligand-binding protein. In various embodiments, the peak emission wavelength of the biosensor shifts by at least about 5, 10, 15, 20, 30, 40, 50, or by about 5-50 nm when the biosensor binds to ligand. In certain embodiments, the emission intensity of the biosensor increases by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, or 300% when the biosensor binds to ligand. In various embodiments, the signal produced by the reporter group persists for at least 1 nanoseconds (ns), 5 ns, 10 ns, 25 ns, 50 ns, 75 ns, 100 ns, 200 ns, 300 ns, 400 ns, 500 ns, 600 ns, 700 ns, 800 ns, 900 ns, 0.001 milliseconds (ms), 0.01 ms, 0.1 ms, 1 ms, 5 ms, 10 ms, 20 ms, 25 ms, 50 ms, 100 ms, or 500 ms when the ligand binds to the ligand-binding protein.
Ligand-Binding Proteins Aspects of the present subject matter provide biosensors comprising a ligand-binding protein that binds a ligand of interest. Non-limiting examples of ligands include sugars (such as glucose, galactose, lactose, arabinose, ribose, and maltose), lactate, urea, anions (e.g., bicarbonate, phosphate, sulfate, and halide anions such as chloride, fluoride, iodide, astatide, ununseptide, and bromide), cations (e.g., calcium, iron, and hydrogen ions), dipeptides, and amino acids (such as histidine, glutamine, glutamate, aspartate).
The ligand-binding protein may comprise a naturally occurring protein or a protein that is modified compared to a naturally occurring protein. For example, the ligand-binding protein may comprise one or more mutations compared to a naturally occurring protein. In some embodiments, the naturally occurring protein is a naturally occurring counterpart of the ligand-binding protein (e.g., the ligand-binding protein is a mutant of the naturally occurring counterpart).
A "naturally occurring counterpart" of a mutant polypeptide is a polypeptide produced in nature from which the mutant polypeptide has been or may be derived (e.g., by one or more mutations). For example, the naturally occurring counterpart is an endogenous polypeptide produced by an organism in nature, wherein the endogenous polypeptide typically does not have one or more of the mutations present in the mutant polypeptide. For convenience and depending on context, a naturally occurring counterpart may be referred to herein for the purpose of comparison and to illustrate the location and/or presence of one or more mutations, binding activities, and/or structural features.
As used herein, a "mutation" is a difference between the amino acid sequence of a modified polypeptide/protein and a naturally occurring counterpart. A
polypeptide having a mutation may be referred to as a "mutant." Non-limiting examples of mutations include insertions, deletions, and substitutions. However, the term "mutation"
excludes (i) the addition of amino acids to the N-terminus or C-terminus of a polypeptide, and (ii) the omission/deletion/replacement of a polypeptide's signal peptide (e.g., replacement with another signal peptide or with a methionine).
The addition of amino acids to the N-terminus or C-terminus of a protein via a peptide bond may be referred to herein as a "fusion" of the amino acids to the protein. Similarly, an exogenous protein fused to amino acids (e.g., another protein, a fragment, a tag, or a polypeptide moiety) at its N-terminus or C-terminus may be referred to as a "fusion protein."
The added amino acids may comprise a non-native polypeptide, e.g., a polypeptide reporter group such as a fluorescent protein, a moiety that facilitates the isolation or modification of a polypeptide, or a moiety that facilitates the attachment of a polypeptide to a substrate or surface. As used herein, "non-native" when referring to the added amino acids (e.g., a "polypeptide") of a fusion protein indicates that the polypeptide is not naturally part of the protein to which it is fused in the fusion protein. For example, the sequence of a non-native polypeptide ("added amino acids") that is fused to a protein is encoded by an organism other than the organism from which the protein is derived, is not known to be naturally encoded by any organism, or is encoded by a gene other than the wild-type gene that encodes an endogenous version of the protein.
As used herein the term "signal peptide" refers to a short (e.g., 5-30 or 10-100 amino acids long) stretch of amino acids at the N-terminus of a protein that directs the transport of the protein. In various embodiments, the signal peptide is cleaved off during the post-translational modification of a protein by a cell. Signal peptides may also be referred to as "targeting signals," "leader sequences," "signal sequences," "transit peptides," or "localization signals." In instances where a signal peptide is not defined for a ligand-binding protein discussed herein, the signal peptide may optionally be considered to be, e.g., the first 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 5-15, 5-20, 5-25, 5-100, 10-15, 10-20, 10-25, 10-50, 10-100, 25-50, 25-75, or 25-100 amino acids from the N-terminus of the translated protein (compared to a protein that has not had the signal peptide removed, e.g., compared to a naturally occurring protein).
In some embodiments, the ligand-binding protein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 1-10, 1-15, 1-20, 5-15, 5-20, 10-25, 10-50, 20-50, 25-75, 25-100 or more mutations compared to a naturally occurring protein while retaining at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5%, or about 100% of the activity of the naturally occurring protein. Mutations include but are not limited to substitutions, insertions, and deletions. Non-limiting examples of ligand-binding proteins may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 1-10, 1-15, 1-20, 5-15, 5-20, 10-25, 10-50, 20-50, 25-75, 25-100, or more substitution mutations compared to a naturally occurring protein while retaining at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5%, or about 100% of the activity of the naturally occurring protein. In embodiments, at least one amino acid of the ligand-binding protein has been substituted with a cysteine.
Alternatively or in addition, a ligand-binding protein may include one or more mutations that remove a cysteine, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more substitutions or deletions of a cysteine compared to a naturally occurring protein.
Alternatively, the ligand-binding protein is not a mutant. For example, a reporter group is fused to the N-terminus or the C-terminus of the ligand-binding protein.
In various embodiments, a ligand-binding protein may comprise a stretch of amino acids (e.g., the entire length of the ligand-binding protein or a portion comprising at least about 50, 100, 200, 250, 300, or 350 amino acids) in a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, or 99.5%
identical to an amino acid sequence of a naturally occurring protein.
In some embodiments, the mutations are conservative, and the present subject matter includes many ligand-binding proteins in which the only mutations are substitution mutations. In non-limiting examples, a ligand-binding protein has no deletions or insertions compared to a naturally occurring protein (e.g., a naturally occurring counterpart).
Alternatively, a ligand-binding protein may have (i) less than about 5, 4, 3, 2, or 1 inserted amino acids, and/or (ii) less than about 5, 4, 3, 2, or 1 deleted amino acids compared to a naturally occurring protein.

In various embodiments, a naturally occurring protein to which a ligand-binding protein is compared or has been derived (e.g., by mutation, fusion, or other modification) from a prokaryotic ligand-binding protein such as a bacterial ligand-binding protein. For example, the prokaryotic ligand-binding protein is a mutant, fragment, or variant of a natural (i.e., wild-type) bacterial protein. In various embodiments, the bacterial ligand-binding protein is from a thermophilic, mesophilic, or cryophilic prokaryotic microorganism (e.g., a thermophilic, mesophilic, or cryophilic bacterium).
A microorganism is "thermophilic" if it is capable of surviving, growing, and reproducing at temperatures between 41 and 140 C (106 and 284 F), inclusive.
In various embodiments, a thermophilic organism has an optimal growth temperature between 41 and 140 C, or that is at least about 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, or 140 C. Many thermophiles are archaea. Thermophilic eubacteria are suggested to have been among the earliest bacteria. Thermophiles are found in various geothermally heated regions of the Earth, such as hot springs and deep sea hydrothermal vents, as well as decaying plant matter, such as peat bogs and compost. Unlike other types of microorganisms, thermophiles can survive at much hotter temperatures, whereas other bacteria would be damaged and sometimes killed if exposed to the same temperatures.
Thermophiles may be classified into three groups: (1) obligate thermophiles;
(2) facultative thermophiles; and (3) hyperthermophiles. Obligate thermophiles (also called extreme thermophiles) require such high temperatures for growth, whereas facultative thermophiles (also called moderate thermophiles) can thrive at high temperatures, but also at lower temperatures (e.g. below 50 C). Hyperthermophiles are particularly extreme thermophiles for which the optimal temperatures are above 80 C. Some microorganisms can live at temperatures higher than 100 C at large depths in the ocean where water does not boil because of high pressure. Many hyperthermophiles are also able to withstand other environmental extremes such as high acidity or radiation levels. A compound (e.g., a protein or biosensor) is "thermotolerant" if it is capable of surviving exposure to temperatures above 41 C. For example, in some embodiments a thermotolerant biosensor retains its function and does not become denatured when exposed to a temperature of about 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, or 140 C for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30 or more minutes. In some embodiments, the thermotolerant compound survives exposure to 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, or 140 C under pressure.

A microorganism is "mesophilic" if it is capable of surviving, growing, and reproducing at temperatures between 20 and 40 C (68 and 104 F), inclusive.
"Psychrophiles" or "cryophiles" are microorganisms that are capable of growth and reproduction in cold temperatures. In various embodiments, a psychrophile is capable of growth and reproduction at a temperature of 10 C or less, e.g., between ¨20 C
and +10 C.
In some embodiments, the microbial protein is produced by a bacterial microorganism, an archaean microorganism, an algal microorganism, a protozoan microorganism, or a fungal microorganism. In non-limiting examples, the microbial protein is produced by a Gram-positive bacterium or a Gram-negative bacterium. In various embodiments, a biosensor comprises a modified (e.g., mutated, fused, and/or conjugated) periplasmic binding protein or a cytoplasmic binding protein.
Aspects of the present subject matter provide a ligand-binding protein with a mutation that alters the interaction of the ligand-binding protein with a ligand. For example, the ligand-binding protein comprises a mutation that alters the interaction of the ligand-binding protein with the ligand compared to a naturally occurring counterpart. In some embodiments, the ligand-binding protein comprises a mutation that alters the interaction of an amino acid of the ligand-binding protein with a water molecule compared to a naturally occurring counterpart.
In some embodiments, the ligand-binding protein does not comprise a signal peptide.
For example, the signal peptide (e.g., that is present in a naturally occurring counterpart) may be replaced with a methionine.
Exemplary implementations relate to a ligand such as sugars (such as glucose, galactose, lactose, arabinose, ribose, and maltose), lactate, urea, anions (e.g., chloride, bicarbonate, phosphate, and sulfate), cations (e.g., calcium and iron), dipeptides, amino acids (such as histidine, glutamine, glutamate, and aspartate). For example, the biosensor may comprise a mutant of, a fragment of, or a fusion protein comprising a microbial ligand-binding protein. In embodiments, the ligand-binding protein is not a mutant or fragment to which a non-native polypeptide has been attached or added.
The ratiometric reagentless biosensors produce precise measurements over extended concentration ranges, e.g. from 0.0001 mM to 100 mM, in sample volumes of less than about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 1.1.1. In some embodiments, the ligand-binding protein comprises a mutation that alters (e.g., increases or decreases) the interaction of the mutant with bound ligand compared to a naturally occurring protein (e.g., a microbial ligand-binding protein). In non-limiting examples, the ligand-binding protein comprises a mutation that alters (e.g., increases or decreases) the mutant's affinity and/or specificity for ligand compared to a unmutated ligand-binding protein (e.g., a microbial ligand-binding protein).
In certain embodiments, the ligand-binding protein comprises a mutation that alters the interaction between the protein and bound ligand, a mutation that alters the equilibrium between the open and closed states of the ligand-binding protein, a mutation that alters the interaction between the ligand-binding protein and a reporter group (such as a fluorescent conjugate, e.g., the interaction with a carbonyl group or a naphthalene ring of a prodan-derived fluorophore such as Acrylodan or Badan), and/or a mutation that impacts indirect interactions that alter the geometry of the ligand binding site. In various embodiments, the mutation does not reduce, or negligibly impacts, the thermostability of the ligand-binding protein. In some embodiments, the mutation alters the thermostability of the ligand-binding protein by less than about 1, 2, 3, 4, 5, or 10 C.
The present subject matter provides a glucose-galactose binding protein GGBP
that is or is a mutant of: an Escherichia sp. (e.g., E. albertii, E. coli, E.
fergusonii, E. hermannii, or E. vulneris) GGBP; a Thermoanaerobacter sp. (e.g., T. acetoethylicus, T.
brockii, T.
ethanolicus, T. italicus, T. kivui, T. mathranii, T. pseudethanolicus, T.
siderophilus, T.
sulfurigignens, T. sulfurophilus, T. thermocopriae, T. thermohydrosulfuricus, T.
thermosaccharolyticum, T. uzonensis, or T. wiegelii) GGBP; a Clostridium sp.
(e.g., C.
absonum, C. aceticum, C. acetireducens, C. acetobutylicum, C. acidisoli, C.
aciditolerans, C.
acidurici, C. aerotolerans, C. aestuarii, C. akagii, C. aldenense, C.
aldrichii, C. algidicarni, C. algidixylanolyticum, C. algifaecis, C. algoriphilum, C. alkalicellulosi, C.
aminophilum, C.
aminovalericum, C. amygdalinum, C. amylolyticum, C. arbusti, C. arcticum, C.
argentinense, C. asparagiforme, C. aurantibutyricum, C. autoethanogenum, C. baratii, C.
barkeri, C.
bartlettii, C. beijerinckii, C. bifermentans, C. bolteae, C. bornimense, C.
botulinum, C.
bowmanii, C. bryantii, C. butyricum, C. cadaveris, C. caenicola, C.
caminithermale, C.
carboxidivorans, C. carnis, C. cavendishii, C. celatum, C. celerecrescens, C.
cellobioparum, C. cellulofermentans, C. cellulolyticum, C. cellulosi, C. cellulovorans, C.
chartatabidum, C.
chauvoei, C. chromiireducens, C. citroniae, C. clariflavum, C.
clostridioforme, C. coccoides, C. cochlearium, C. colletant, C. colicanis, C. colinum, C. collagenovorans, C.
cylindrosporum, C. difficile, C. diolis, C. disporicum, C. drakei, C. durum, C. estertheticum, C. estertheticum estertheticum, C. estertheticum laramiense, C. fallax, C.
felsineum, C.
fervidum, C. fimetarium, C. formicaceticum, C. frigidicarnis, C. frigoris, C.
ganghwense, C.

gasigenes, C. ghonii, C. glycolicum, C. glycyrrhizinilyticum, C. grantii, C.
haemolyticum, C.
halophilum, C. hastiforme, C. hathewayi, C. herbivorans, C. hiranonis, C.
histolyticum, C.
homopropionicum, C. huakuii, C. hungatei, C. hydrogeniformans, C.
hydroxybenzoicum, C.
hylemonae, C. jejuense, C. indolis, C. innocuum, C. intestinale, C.
irregulare, C. isatidis, C.
josui, C. kluyveri, C. lactatifermentans, C. lacusfryxellense, C. laramiense, C. lavalense, C.
lentocellum, C. lentoputrescens, C. leptum, C. limosum, C. litorale, C.
lituseburense, C.
ljungdahlii, C. lortetii, C. lundense, C. magnum, C. malenominatum, C.
mangenotii, C.
mayombei, C. methoxybenzovorans, C. methylpentosum, C. neopropionicum, C.
nexile, C.
nitrophenolicum, C. novyi, C. oceanicum, C. orbiscindens, C. oroticum, C.
oxalicum, C.
papyrosolvens, C. paradoxum, C. paraperfringens, C. paraputrificum, C. pascui, C.
pasteurianum, C. peptidivorans, C. perenne, C. perfringens, C. pfennigii, C.
phytofermentans, C. piliforme, C. polysaccharolyticum, C. populeti, C.
propionicum, C.
proteoclasticum, C. proteolyticum, C. psychrophilum, C. puniceum, C.
purinilyticum, C.
putrefaciens, C. putrificum, C. quercicolum, C. quinii, C. ramosum, C. rectum, C. roseum, C.
saccharobutylicum, C. saccharogumia, C. saccharolyticum, C.
saccharoperbutylacetonicum, C. sardiniense, C. sartagoforme, C. scatologenes, C. schirmacherense, C.
scindens, C.
septicum, C. sordellii, C. sphenoides, C. spiroforme, C. sporogenes, C.
sporosphaeroides, C.
stercorarium, C. stercorarium leptospartum, C. stercorarium stercorarium, C.
stercorarium thermolacticum, C. sticklandii, C. straminisolvens, C. subterminale, C.
sufflavum, C.
sulfidigenes, C. symbiosum, C. tagluense, C. tepidiprofundi, C. termitidis, C.
tertium, C.
tetani, Clostridium tetanomorphum, C. thermaceticum, C. thermautotrophicum, C.

thermoalcaliphilum, C. thermobutyricum, C. thermocellum, C. thermocopriae, C.
thermohydrosulfuricum, C. thermolacticum, C. thermopalmarium, C.
thermopapyrolyticum, C. thermosaccharolyticum, C. thermosuccinogenes, C. thermosulfurigenes, C.
thiosulfatireducens, C. tyrobutyricum, C. uliginosum, C. ultunense, C.
villosum, C. vincentii, C. viride, C. xylanolyticum, or C. xylanovorans) GGBP; a Salmonella sp. [e.g., S. bongori, S.
enterica, S. enterica subspecies enterica, S. enterica subspecies salamae, S.
enterica subspecies arizonae, S. enterica subspecies diarizonae, S. enterica subspecies houtenae, S.
enterica subspecies indica, or S. enterica subspecies enterica serovar Typhimurium (S.
typhimurium)] GGBP; a Caldicellulosiruptor sp. (e.g., C. saccharolyticus, C.
acetigenus, C.
bescii, C. changbaiensis, C. hydrothermalis, Caldicellulosiruptor hydrother, C.
kristjanssonii, C. kronotskyensis, C. lactoaceticus, C. owensensis, or C.
obsidiansis) GGBP; a Paenibacillus sp. (e.g., P. agarexedens, P. agaridevorans, P. alginolyticus, P. alkaliterrae, P.

alvei, P. amylolyticus, P. anaericanus, P. antarcticus, P. assamensis, P.
azoreducens, P.
azotofixans, P. barcinonensis, P. borealis, P. brasilensis, P. brassicae, P.
campinasensis, P.
chinjuensis, P. chitinolyticus, P. chondroitinus, P. cineris, P. cookii, P.
curdlanolyticus, P.
daejeonensis, P. dendritiformis, P. durum, P. ehimensis, P. elgii, P.
favisporus, P.
glucanolyticus, P. glycanilyticus, P. gordonae, P. graminis, P. granivorans, P. hodogayensis, P. illinoisensis, P. jamilae, P. kobensis, P. koleovorans, P. koreensis, P.
kribbensis, P. lactis, P. larvae, P. lautus, P. lentimorbus, P. macerans, P. macquariensis, P.
massiliensis, P.
mendelii, P. motobuensis, P. naphthalenovorans, P. nematophilus, P. odorifer, P. pabuli, P.
peoriae, P. phoenicis, P. phyllosphaerae, P. polymyxa, P. popilliae, P.
pulvifaciens, P.
rhizosphaerae, P. sanguinis, P. stellifer, P. terrae, P. thiaminolyticus, P.
timonensis, P.
tylopili, P. turicensis, P. validus, P. vortex, P. vulneris, P. wynnii, P.
xylanilyticus) GGBP; a Butyrivibrio sp. (e.g., B. proteoclasticus, B. crossotus, B. fibrisolvens, or B. hungatei) GGBP;
a Roseburia sp. (e.g., R. intestinalis, R. faecis, R. hominis, or R.
inulinivorans) GGBP; a Faecalibacterium sp. (e.g., F. prausnitzii) GGBP; an Erysipelothrix sp. (e.g., E.
rhusiopathiae, E. inopinata, or E. tonsillarum) GGBP; or an Eubacterium sp.
(e.g., E. rectale, E. acidaminophilum, E. nodatum, E. oxidoreducens, or E. foedans) GGBP.
The present subject matter provides a urea-binding protein that is or is a mutant of: an Marinomas sp. (e.g., M posidonica ) urea-binding protein; a Marinobacter sp.
(e.g., M
adhaerens, M algicola, M alkaliphilus, M antarcticus, M arcticus, Maromaticivorans, M
bryozoorum, M daepoensis, M daqiaonensis, M excellens, M. flavimaris, M
gudaonensis, M guineae, M halophilus, M gudaonensis, M hydrocarbonoclasticus, M koreensis, M
lacisalsi, M lipolyticus, M litoralis, M lutaoensis, M maritimus, M mobilis, M

nitratireducens, M oulmenensis, M pelagius, M persicus, M psychrophilus, M
nanhaiticus, M salarius, M salicampi, M salsuginis, M santoriniensis, M sediminum, M
segnicrescens, M shengliensis, M squalenivorans, M similis, M szutsaonensis, M vinifirmus, M
xestospongiae, M zhanjiangensis, or M zhejiangensis) urea-binding protein; a Bacillus sp.
(e.g., B. acidiceler, B. acidicola, B. acidiproducens, B. acidocaldarius, B.
acidoterrestris, B.
aeolius, B. aerius, B. aerophilus, B. agaradhaerens, B. agri, B. aidingensis, B. akibai, B.
alcalophilus, B. algicola, B. alginolyticus, B. alkalidiazotrophicus, B.
alkalinitrilicus, B.
alkalisediminis, B. alkalitelluris, B. altitudinis, B. alveayuensis, B. alvei, B.
amyloliquefaciens, B. a. subsp. amyloliquefaciens, B. a. subsp. plantarum, B.
amylolyticus, B.
andreesenii, B. aneurinilyticus, B. anthracis, B. aquimaris, B. arenosi, B.
arseniciselenatis, B. arsenicus, B. aurantiacus, B. arvi, B. aryabhattai, B. asahii, B.
atrophaeus, B.

axarquiensis, B. azotofixans, B. azotoformans, B. badius, B. barbaricus, B.
bataviensis, B.
beijingensis, B. benzoevorans, B. beringensis, B. berkeleyi, B. beveridgei, B.
bogoriensis, B.
boroniphilus, B. borstelensis, B. brevis Migula, B. butanolivorans, B.
canaveralius, B.
carboniphilus, B. cecembensis, B. cellulosilyticus, B. centrosporus, B.
cereus, B.
chagannorensis, B. chitinolyticus, B. chondroitinus, B. choshinensis, B.
chungangensis, B.
cibi, B. circulans, B. clarkii, B. clausii, B. coagulans, B. coahuilensis, B.
cohnii, B. composti, B. curdlanolyticus, B. cycloheptanicus, B. cytotoxicus, B. daliensis, B.
decisifrondis, B.
decolorationis, B. deserti, B. dipsosauri, B. drentensis, B. edaphicus, B.
ehimensis, B.
eiseniae, B. enclensis, B. endophyticus, B. endoradicis, B. farraginis, B.
fastidiosus, B.
fengqiuensis, B. firmus, B. flexus, B. foraminis, B. fordii, B. formosus, B.
fortis, B. fumarioli, B. funiculus, B. fusiformis, B. galactophilus, B. galactosidilyticus, B.
galliciensis, B. gelatini, B. gibsonii, B. ginsengi, B. ginsengihumi, B. ginsengisoli, B. globisporus, B.
g. subsp.
globisporus, B. g. subsp. marinus, B. glucanolyticus, B. gordonae, B.
gottheilii, B. graminis, B. halmapalus, B. haloalkaliphilus, B. halochares, B. halodenitrificans, B.
halodurans, B.
halophilus, B. halosaccharovorans, B. hemicellulosilyticus, B. hemicentroti, B.
herbersteinensis, B. horikoshii, B. horneckiae, B. horti, B. huizhouensis, B.
humi, B.
hwajinpoensis, B. idriensis, B. indicus, B. infantis, B. infernus, B.
insolitus, B. invictae, B.
iranensis, B. isabeliae, B. isronensis, B. jeotgali, B. kaustophilus, B.
kobensis, B. kochii, B.
kokeshiiformis, B. koreensis, B. korlensis, B. kribbensis, B. kndwichiae, B.
laevolacticus, B.
larvae, B. laterosporus, B. lautus, B. lehensis, B. lentimorbus, B. lentus, B.
licheniformis, B.
ligniniphilus, B. litoralis, B. locisalis, B. luciferensis, B. luteolus, B.
luteus, B. macauensis, B.
macerans, B. macquariensis, B. macyae, B. malacitensis, B. mannanilyticus, B.
marisflavi, B.
marismortui, B. marmarensis, B. massiliensis, B. megaterium, B. mesonae, B.
methanolicus, B. methylotrophicus, B. migulanus, B. mojavensis, B. mucilaginosus, B.
muralis, B.
murimartini, B. mycoides, B. naganoensis, B. nanhaiensis, B. nanhaiisediminis, B. nealsonii, B. neidei, B. neizhouensis, B. niabensis, B. niacini, B. novalis, B.
oceanisediminis, B.
odysseyi, B. okhensis, B. okuhidensis, B. oleronius, B. oryzaecorticis, B.
oshimensis, B.
pabuli, B. pakistanensis, B. pallidus, B. pallidus, B. panacisoli, B.
panaciterrae, B.
pantothenticus, B. parabrevis, B. paraflexus, B. pasteurii, B. patagoniensis, B. peoriae, B.
persepolensis, B. persicus, B. pervagus, B. plakortidis, B. pocheonensis, B.
polygoni, B.
polymyxa, B. popilliae, B. pseudalcalophilus, B. pseudofirmus, B.
pseudomycoides, B.
psychrodurans, B. psychrophilus, B. psychrosaccharolyticus, B.
psychrotolerans, B.
pulvifaciens, B. pumilus, B. purgationiresistens, B. pycnus, B. qingdaonensis, B. qingshengii, B. reuszeri, B. rhizosphaerae, B. rigui, B. ruris, B. safensis, B. salarius, B. salexigens, B.
saliphilus, B. schlegelii, B. sediminis, B. selenatarsenatis, B.
selenitireducens, B.
seohaeanensis, B. shacheensis, B. shackletonii, B. siamensis, B. silvestris, B. simplex, B.
siralis, B. smithii, B. soli, B. solimangrovi, B. solisalsi, B. songklensis, B. sonorensis, B.
sphaericus, B. sporothermodurans, B. stearothermophilus, B. stratosphericus, B.
subterraneus, B. subtilis, B. s. subsp. inaquosorum, B. s. subsp. spizizenii, B. s. subsp.
subtilis, B. taeanensis, B. tequilensis, B. thermantarcticus, B.
thermoaerophilus, B.
thermoamylovorans, B. thermocatenulatus, B. thermocloacae, B. thermocopriae, B.
thermodenitrificans, B. thermoglucosidasius, B. thermolactis, B.
thermoleovorans, B.
thermophilus, B. thermoruber, B. thermosphaericus, B. thiaminolyticus, B.
thioparans, B.
thuringiensis, B. tianshenii, B. trypoxylicola, B. tusciae, B. validus, B.
vallismortis, B.
vedderi, B. velezensis, B. vietnamensis, B. vireti, B. vulcani, B. wakoensis, B.
weihenstephanensis, B. xiamenensis, B. xiaoxiensis, or B. zhanjiangensis) urea-binding protein; a Desulfotomaculum sp. (e.g., D. ruminis, D. nigrificans, D.
australicum, D.
thermobenzoicum, D. geothermicum, D. thermocisternum, D. aeronauticum, D.
halophilum, D. kuznetsovii, D. thermoacetoxidans, D. thermosapovorans, D. acetoxidans, D.
reducens, D.
putei, D. luciae, D. gibsoniae, D. sapomandens, D. alkaliphilum, D. sp. FSB6, D. sp. ASRB-Zg, D. sp. 175,D. sp. 176,D. sp. 171,D. sp. C40-3,D. sp. TPOSR, D. sp. WW1, D.
sp.
SRB-M, D. sp. Mechichi-2001, D. solfataricum, D. sp. ECP-05, D. sp. MPNegl, D.
sp.
0x39, D. sp. RL50L1, D. alcoholivorax, D. sp. NC402, D. sp. NB401, D. sp.
NA401, D.
salinum, D. carboxydivorans, D. arcticum, D. thermosubterraneum, D. indicum, D. sp. Lac2, D. sp. CYP1, D. sp. CYP9, D. sp. IS3205, D. sp. Srb55, D. sp. Iso-W2, D. sp.
2, D.
hydrothermale, D. sp. ADR22, D. sp. Hbr7, D. sp. J 175, D. sp. J 176, D. sp.
DSM 7440, D. sp. DSM 7474, D. sp. DSM 7475, D. sp. DSM 7476, D. sp. DSM 8775, D. sp. cs1-2, or D.
sp. MJ1) urea-binding protein; a Geobacillus sp. (e.g., G.
thermoglucosidasius, G.
stearothermophilus, G. jurassicus, G. toebii) urea-binding protein; a Clostridium sp. (e.g., C.
absonum, C. aceticum, C. acetireducens, C. acetobutylicum, C. acidisoli, C.
aciditolerans, C.
acidurici, C. aerotolerans, C. aestuarii, C. akagii, C. aldenense, C.
aldrichii, C. algidicarni, C. algidixylanolyticum, C. algifaecis, C. algoriphilum, C. alkalicellulosi, C.
aminophilum, C.
aminovalericum, C. amygdalinum, C. amylolyticum, C. arbusti, C. arcticum, C.
argentinense, C. asparagiforme, C. aurantibutyricum, C. autoethanogenum, C. baratii, C.
barkeri, C.
bartlettii, C. beijerinckii, C. bifermentans, C. bolteae, C. bornimense, C.
botulinum, C.
bowmanii, C. bryantii, C. butyricum, C. cadaveris, C. caenicola, C.
caminithermale, C.

carboxidivorans, C. carnis, C. cavendishii, C. celatum, C. celerecrescens, C.
cellobioparum, C. cellulofermentans, C. cellulolyticum, C. cellulosi, C. cellulovorans, C.
chartatabidum, C.
chauvoei, C. chromiireducens, C. citroniae, C. clariflavum, C.
clostridioforme, C. coccoides, C. cochlearium, C. colletant, C. colicanis, C. colinum, C. collagenovorans, C.
cylindrosporum, C. difficile, C. diolis, C. disporicum, C. drakei, C. durum, C. estertheticum, C. estertheticum estertheticum, C. estertheticum laramiense, C. fallax, C.
felsineum, C.
fervidum, C. fimetarium, C. formicaceticum, C. frigidicarnis, C. frigoris, C.
ganghwense, C.
gasigenes, C. ghonii, C. glycolicum, C. glycyrrhizinilyticum, C. grantii, C.
haemolyticum, C.
halophilum, C. hastiforme, C. hathewayi, C. herbivorans, C. hiranonis, C.
histolyticum, C.
homopropionicum, C. huakuii, C. hungatei, C. hydrogeniformans, C.
hydroxybenzoicum, C.
hylemonae, C. jejuense, C. indolis, C. innocuum, C. intestinale, C.
irregulare, C. isatidis, C.
josui, C. kluyveri, C. lactatifermentans, C. lacusfryxellense, C. laramiense, C. lavalense, C.
lentocellum, C. lentoputrescens, C. leptum, C. limosum, C. litorale, C.
lituseburense, C.
ljungdahlii, C. lortetii, C. lundense, C. magnum, C. malenominatum, C.
mangenotii, C.
mayombei, C. methoxybenzovorans, C. methylpentosum, C. neopropionicum, C.
nexile, C.
nitrophenolicum, C. novyi, C. oceanicum, C. orbiscindens, C. oroticum, C.
oxalicum, C.
papyrosolvens, C. paradoxum, C. paraperfringens, C. paraputrificum, C. pascui, C.
pasteurianum, C. peptidivorans, C. perenne, C. perfringens, C. pfennigii, C.
phytofermentans, C. piliforme, C. polysaccharolyticum, C. populeti, C.
propionicum, C.
proteoclasticum, C. proteolyticum, C. psychrophilum, C. puniceum, C.
purinilyticum, C.
putrefaciens, C. putrificum, C. quercicolum, C. quinii, C. ramosum, C. rectum, C. roseum, C.
saccharobutylicum, C. saccharogumia, C. saccharolyticum, C.
saccharoperbutylacetonicum, C. sardiniense, C. sartagoforme, C. scatologenes, C. schirmacherense, C.
scindens, C.
septicum, C. sordellii, C. sphenoides, C. spiroforme, C. sporogenes, C.
sporosphaeroides, C.
stercorarium, C. stercorarium leptospartum, C. stercorarium stercorarium, C.
stercorarium thermolacticum, C. sticklandii, C. straminisolvens, C. subterminale, C.
suffiavum, C.
sulfidigenes, C. symbiosum, C. tagluense, C. tepidiprofundi, C. termitidis, C.
tertium, C.
tetani, Clostridium tetanomorphum, C. thermaceticum, C. thermautotrophicum, C.

thermoalcaliphilum, C. thermobutyricum, C. thermocellum, C. thermocopriae, C.
thermohydrosulfuricum, C. thermolacticum, C. thermopalmarium, C.
thermopapyrolyticum, C. thermosaccharolyticum, C. thermosuccinogenes, C. thermosulfurigenes, C.
thiosulfatireducens, C. tyrobutyricum, C. uliginosum, C. ultunense, C.
villosum, C. vincentii, C. viride, C. xylanolyticum, or C. xylanovorans) urea-binding protein; a Caldicellulosiruptor sp. (e.g., C. acetigenus, C. bescii, C. changbaiensis, C. hydrothermalis, C.
kristjanssonii, C.
kronotskyensis, C. lactoaceticus, C. owensensis, or C. saccharolyticus) urea-binding protein;
a Thermocrinis sp. (e.g., T. ruber, T. albus, or T. minervae) urea-binding protein; a Synechoccus sp. (e.g., S. ambiguus, S. arcuatus var. cakicolus, S.
bigranulatus, S.
brunneolus S. caldarius, S. capitatus, S. carcerarius, S. elongatus, S.
endogloeicus, S.
epigloeicus, S. ferrunginosus, S. intermedius, S. koidzumii, S. lividus, S.
marinus, S.
minutissimus, S. mundulus, S. nidulans, S. rayssae, S. rhodobaktron, S. roseo-persicinus, S.
roseo-purpureus, S. salinarum, S. salinus, S. sciophilus, S. sigmoideus, S.
spongiarum, S.
subsalsus, S. sulphuricus, S. vantieghemii, S. violaceus, S. viridissimus, or S. vtdcanus) urea-binding protein; a Paenibacillus sp. (e.g., P. agarexedens, P. agaridevorans, P. alginolyticus, P. alkaliterrae, P. alvei, P. amylolyticus, P. anaericanus, P. antarcticus, P.
assamensis, P.
azoreducens, P. azotofixans, P. barcinonensis, P. borealis, P. brasilensis, P.
brassicae, P.
campinasensis, P. chinjuensis, P. chitinolyticus, P. chondroitinus, P.
cineris, P. cookii, P.
curdlanolyticus, P. daejeonensis, P. dendritiformis, P. durum, P. ehimensis, P. elgii, P.
favisporus, P. glucanolyticus, P. glycanilyticus, P. gordonae, P. graminis, P.
granivorans, P.
hodogayensis, P. illinoisensis, P. jamilae, P. kobensis, P. koleovorans, P.
koreensis, P.
kribbensis, P. lactis, P. larvae, P. lautus, P. lentimorbus, P. macerans, P.
macquariensis, P.
massiliensis, P. mendelii, P. motobuensis, P. naphthalenovorans, P.
nematophilus, P.
odorifer, P. pabuli, P. peoriae, P. phoenicis, P. phyllosphaerae, P. polymyxa, P. popilliae, P.
pulvifaciens, P. rhizosphaerae, P. sanguinis, P. stellifer, P. terrae, P.
thiaminolyticus, P.
timonensis, P. tylopili, P. turicensis, P. validus, P. vortex, P. vulneris, P.
wynnii, P.
xylanilyticus) urea-binding protein; or a Thermosynechococcus sp. (e.g., T.
elongatus or T.
vulcanus) urea-binding protein.
The present subject matter provides a glucose-binding protein that is or is a mutant of:
an Thermus sp. (e.g., T. caldophilus, T. eggertssonii, T. kawarayensis, T.
murrieta, T.
nonproteolyticus, T. parvatiensis, T. rehai, T. yunnanensis, T.
amyloliquefaciens, T.
antranikianii, T. aquaticus, T. arciformis, T. brockianus, T. caliditerrae, T.
chliarophilus, T.
composti, T. filiformis, T. igniterrae, T. islandicus, T. oshimai, T.
profundus, T. scotoductus, T. tengchongensis, or T. thermophilus) glucose-binding protein; a Deinococcus sp. (e.g., D.
aquivivus, D. puniceus, D. soli, D. xibeiensis, D. aerius, D. aerolatus, D.
aerophilus, D.
aetherius, D. alpinitundrae, D. altitudinis, D. apachensis, D. aquaticus, D.
aquatilis, D.
aquiradiocola, D. caeni, D. cellulosilyticus, D. claudionis, D. daejeonensis, D.
depolymerans, D. deserti, D. erythromyxa, D. ficus, D. frigens, D.
geothermalis, D.

gobiensis, D. grandis, D. hohokamensis, D. hopiensis, D. indicus, D.
maricopensis, D.
marmoris, D. metalli, D. misasensis, D. murrayi, D. navajonensis, D.
papagonensis, D.
peraridilitoris, D. pimensis, D. piscis, D. proteolyticus, D. radiodurans, D.
radiomollis, D.
radiophilus, D. radiopugnans, D. reticulitermitis, D. roseus, D. saxicola, D.
sonorensis, D.
wulumuqiensis, D. xibeiensis, D. xinjiangensis, D. yavapaiensis, or D.
yunweiensis) glucose-binding protein; a Thermotoga sp. (e.g., T. caldifontis, T. elfii, T. hypogea, T. lettingae, T.
maritima, T. naphthophila, T. neapolitana, T. petrophila, T. profunda, T.
subterranea, or T.
thermarum) glucose-binding protein; a Kosmotoga sp. (e.g., K olearia, K
arenicorallina, K
pacifica, or K shengliensis) glucose-binding protein; a Bacillus sp. (e.g., B.
acidiceler, B.
acidicola, B. acidiproducens, B. acidocaldarius, B. acidoterrestris, B.
aeolius, B. aerius, B.
aerophilus, B. agaradhaerens, B. agri, B. aidingensis, B. akibai, B.
alcalophilus, B. algicola, B. alginolyticus, B. alkalidiazotrophicus, B. alkalinitrilicus, B.
alkalisediminis, B.
alkalitelluris, B. altitudinis, B. alveayuensis, B. alvei, B.
amyloliquefaciens, B. a. subsp.
amyloliquefaciens, B. a. subsp. plantarum, B. amylolyticus, B. andreesenii, B.
aneurinilyticus, B. anthracis, B. aquimaris, B. arenosi, B. arseniciselenatis, B. arsenicus, B.
aurantiacus, B. arvi, B. aryabhattai, B. asahii, B. atrophaeus, B.
axarquiensis, B. azotofixans, B. azotoformans, B. badius, B. barbaricus, B. bataviensis, B. beijingensis, B.
benzoevorans, B. beringensis, B. berkeleyi, B. beveridgei, B. bogoriensis, B. boroniphilus, B. borstelensis, B.
brevis Migula, B. butanolivorans, B. canaveralius, B. carboniphilus, B.
cecembensis, B.
cellulosilyticus, B. centrosporus, B. cereus, B. chagannorensis, B.
chitinolyticus, B.
chondroitinus, B. choshinensis, B. chungangensis, B. cibi, B. circulans, B.
clarkii, B. clausii, B. coagulans, B. coahuilensis, B. cohnii, B. composti, B. curdlanolyticus, B.
cycloheptanicus, B. cytotoxicus, B. daliensis, B. decisifrondis, B. decolorationis, B. deserti, B. dipsosauri, B.
drentensis, B. edaphicus, B. ehimensis, B. eiseniae, B. enclensis, B.
endophyticus, B.
endoradicis, B. farraginis, B. fastidiosus, B. fengqiuensis, B. firmus, B.
flexus, B. foraminis, B. fordii, B. formosus, B. fortis, B. fumarioli, B. funiculus, B. fusiformis, B. galactophilus, B.
galactosidilyticus, B. galliciensis, B. gelatini, B. gibsonii, B. ginsengi, B.
ginsengihumi, B.
ginsengisoli, B. globisporus, B. g. subsp. globisporus, B. g. subsp. marinus, B.
glucanolyticus, B. gordonae, B. gottheilii, B. graminis, B. halmapalus, B.
haloalkaliphilus, B.
halochares, B. halodenitrificans, B. halodurans, B. halophilus, B.
halosaccharovorans, B.
hemicellulosilyticus, B. hemicentroti, B. herbersteinensis, B. horikoshii, B.
horneckiae, B.
horti, B. huizhouensis, B. humi, B. hwajinpoensis, B. idriensis, B. indicus, B. infantis, B.
infernus, B. insolitus, B. invictae, B. iranensis, B. isabeliae, B.
isronensis, B. jeotgali, B.

kaustophilus, B. kobensis, B. kochii, B. kokeshiiformis, B. koreensis, B.
korlensis, B.
kribbensis, B. kndwichiae, B. laevolacticus, B. larvae, B. laterosporus, B.
lautus, B. lehensis, B. lentimorbus, B. lentus, B. licheniformis, B. ligniniphilus, B. litoralis, B. locisalis, B.
luciferensis, B. luteolus, B. luteus, B. macauensis, B. macerans, B.
macquariensis, B. macyae, B. malacitensis, B. mannanilyticus, B. marisflavi, B. marismortui, B.
marmarensis, B.
massiliensis, B. megaterium, B. mesonae, B. methanolicus, B. methylotrophicus, B.
migulanus, B. mojavensis, B. mucilaginosus, B. muralis, B. murimartini, B.
mycoides, B.
naganoensis, B. nanhaiensis, B. nanhaiisediminis, B. nealsonii, B. neidei, B.
neizhouensis, B.
niabensis, B. niacini, B. novalis, B. oceanisediminis, B. odysseyi, B.
okhensis, B. okuhidensis, B. oleronius, B. oryzaecorticis, B. oshimensis, B. pabuli, B. pakistanensis, B. pallidus, B.
pallidus, B. panacisoli, B. panaciterrae, B. pantothenticus, B. parabrevis, B.
paraflexus, B.
pasteurii, B. patagoniensis, B. peoriae, B. persepolensis, B. persicus, B.
pervagus, B.
plakortidis, B. pocheonensis, B. polygoni, B. polymyxa, B. popilliae, B.
pseudalcalophilus, B.
pseudofirmus, B. pseudomycoides, B. psychrodurans, B. psychrophilus, B.
psychrosaccharolyticus, B. psychrotolerans, B. pulvifaciens, B. pumilus, B.
purgationiresistens, B. pycnus, B. qingdaonensis, B. qingshengii, B. reuszeri, B.
rhizosphaerae, B. rigui, B. ruris, B. safensis, B. salarius, B. salexigens, B.
saliphilus, B.
schlegelii, B. sediminis, B. selenatarsenatis, B. selenitireducens, B.
seohaeanensis, B.
shacheensis, B. shackletonii, B. siamensis, B. silvestris, B. simplex, B.
siralis, B. smithii, B.
soli, B. solimangrovi, B. solisalsi, B. songklensis, B. sonorensis, B.
sphaericus, B.
sporothermodurans, B. stearothermophilus, B. stratosphericus, B. subterraneus, B. subtilis, B. s. subsp. inaquosorum, B. s. subsp. spizizenii, B. s. subsp. subtilis, B.
taeanensis, B.
tequilensis, B. thermantarcticus, B. thermoaerophilus, B. thermoamylovorans, B.
thermocatenulatus, B. thermocloacae, B. thermocopriae, B. thermodenitrificans , B.
thermoglucosidasius, B. thermolactis, B. thermoleovorans, B. thermophilus, B.
thermoruber, B. thermosphaericus, B. thiaminolyticus, B. thioparans, B. thuringiensis, B.
tianshenii, B.
trypoxylicola, B. tusciae, B. validus, B. vallismortis, B. vedderi, B.
velezensis, B.
vietnamensis, B. vireti, B. vulcani, B. wakoensis, B. weihenstephanensis, B.
xiamenensis, B.
xiaoxiensis, or B. zhanjiangensis) glucose-binding protein; a Staphylothermus sp. (e.g., S.
hellenicus or S. marinus) glucose-binding protein; or an Arthrobacter sp.
(e.g., A. agilis, A.
alkaliphilus, A. alpinus, A. antarcticus, A. aurescens, A. bambusae, A.
castelli, A.
chlorophenolicus, A. citreus, A. cryoconiti, A. cryotolerans, A.
crystallopoietes, A. cumminsii, A. cupressi, A. defluvii, A. enclensis, A. flavus, A. gandavensis, A.
globiformis, A.

gyeryongensis, A. halodurans, A. histidinolovorans, A. humicola, A. koreensis, A. liuii, A.
livingstonensis, A. luteolus, A. methylotrophus, A. monumenti, A.
nanjingensis, A.
nasiphocae, A. nicotinovorans, A. nitroguajacolicus, A. oryzae, A. parietis, A. pascens, A.
pigmenti, A. pityocampae, A. psychrochitiniphilus, A. psychrolactophilus, A.
ramosus, A.
rhombi, A. roseus, A. russicus, A. sanguinis, A. soli, A. stackebrandtii, A.
subterraneus, A.
tecti, A. tumbae, A. viscosus, or A. woluwensis) glucose-binding protein.
The present subject matter provides a lactate-binding protein that is or is a mutant of:
a Thermus sp. (e.g., T. caldophilus, T. eggertssonii, T. kawarayensis, T.
murrieta, T.
nonproteolyticus, T. parvatiensis, T. rehai, T. yunnanensis, T.
amyloliquefaciens, T.
antranikianii, T. aquaticus, T. arciformis, T. brockianus, T. caliditerrae, T.
chliarophilus, T.
composti, T. filiformis, T. igniterrae, T. islandicus, T. oshimai, T.
profundus, T. scotoductus, T. shimai, T. tengchongensis, or T. thermophilus) lactate-binding protein, a Thioalkalivibrio sp. (e.g., T. denitrificans, T. halophilus, T. jannaschii, T. nitratireducens, T. nitratis, T.
paradoxus, T. sulfidiphilus, T. thiocyanodenitrificans, T. thiocyanoxidans, and T. versutus) lactate-binding protein, a Roseobacter sp. (e.g., R. dentrificans, R.
litoralis, R. pelophilus, R.
prionitis, R. sp. 14111/A01/004, R. sp. 1922, R. sp. 27-4, R. sp. 3008, R. sp.
38.98, R. sp.
3X/A02/234, R. sp. 4318-8/1,R. sp. 812,R. sp. 8-1,R. sp. ANT8230, R. sp.
AN'T9082, R. sp.
ANT909, R. sp. ANT9234, R. sp. ANT9240, R. sp. ANT9270, R. sp. ANT9274, R. sp.

ANT9276a, R. sp. AN'T9283, R. sp. ARCTIC-P4, R. sp. ARK9990, R. sp. AzwK-3b, R. sp.
AzwLept-lc, R. sp. B09, R. sp. B-1039, R. sp. B11, R. sp. Ber2105, R. sp.
Ber2107, R. sp.
BS36, R. sp. BS90, R. sp. C115, R. sp. C23, R. sp. CCS2, R. sp. COL2P, R. sp.
COLSP, R. sp.
DG1132, R. sp. DG869, R. sp. DG889, R. sp. DG942, R. sp. Do-34, R. sp. DSS-1, R. sp.
DSS-8, R. sp. H264, R. sp. H265, R. sp. H454, R.. HJ105, R. sp. HJ247, R. sp.
HYL-SA-18, R. sp. HZBC52, R. sp. HZDC27, R. sp. HZDC41, R. sp. HZDC42, R. sp. HZDC43, R.
sp.
HZDC7, R. sp. J2W, R. sp. J356, R. sp. J392, R. sp. J483, R. sp. J486, R. sp.
J504, R. sp. J8W, R. sp. JL-126, R. sp. JL-129, R. sp. JL-131, R.. JL-132, R. sp. JL-135, R. sp.
JL-137, R. sp.
JL-351, R. sp. JL985, R. sp. JLN-A020, R. sp. JLN-A036, R. sp. JLN-A530, R.
sp. KAT10, R.
sp. KAT3, R. sp. KT0202a, R. sp. KT0917, R. sp. KT1117, R. sp. LA7, R. sp.
LA9, R. sp.
LOB-8, R. sp. LZXC12, R. sp. LZXC14, R. sp. LZXC15, R. sp. LZXC16, R. sp.
LZXC20, R.
sp. LZXC23, R. sp. LZXC26, R. sp. LZXC29, R. sp. LZXC4, R. sp. LZXC7, R. sp.
MBT21, R. sp. MBT22, R. sp. MED001, R. sp. MED006, R. sp. MED007, R. sp. MED008, R.
sp.
MED193, R. sp. MED24, R. sp. MED26, R. sp. MED61, R. sp. MED6, R. sp. M5I042, R. sp.
NO51, R. sp. NJ5527, R. sp. NT N37, R. sp. 0C-B2-7, R. sp. 0C-C4-20, R. sp. 00-A3-7, R.

sp. 00-C4-10, R. sp. Pht-4, R. sp. PIC-68, R. sp. PRLIST02, R. sp. PRLIST06, R. sp.
PRLISY01, R. sp. PRLISY03, R. sp. QSSC9-8, R. sp. RED15, R. sp. RED1, R. sp.
RED59, R.
sp. RED68, R. sp. RED85, R. sp. S03, R. sp. SC-B2-2,R. sp. SCB28, R. sp.
SCB31, R. sp.
5CB34, R. sp. 5CB48, R. sp. SDBC1, R. sp. SDBC6, R. sp. SFLA13, R. sp. SIO, R.
sp.
5K209-2-6, R. sp. 5KA26, R. sp. 5KA44, R. sp. 5L25, R. sp. S03, R. sp. 5P0804, R. sp.
SY0P1, R. sp. SY0P2, R. sp. TM1035, R. sp. TM1038, R. sp. TM1040, R. sp.
TM1042, R.
sp. TP9, R. sp. UAzPsJAC-lb, R. sp. UAzPsK-5, R. sp. WED10.10, R. sp. WED1.1, R. sp.
WHOI JT-01, R. sp. WHOI JT-08, R. sp. WHOI JT-22, R. sp. WM2, R. sp. Y2, R.
sp. Y3F, R.
sp. YS-57, R. sp. YSCB-1, or R. sp. YSCB-3) lactate-binding protein, a Marinobacter sp.
(e.g., M adhaerens, M algicola, M alkaliphilus, M antarcticus, M arcticus, M.aromaticivorans, M bryozoorum, M daepoensis, M daqiaonensis, M excellens, M
flavimaris, M gudaonensis, M guineae, M halophilus, M gudaonensis, M
hydrocarbonoclasticus, M koreensis, M lacisalsi, M lipolyticus, M litoralis, M
lutaoensis, M maritimus, M mobilis, M nitratireducens, M oulmenensis, M pelagius, M
persicus, M
psychrophilus, M nanhaiticus, M salarius, M salicampi, M salsuginis, M
santoriniensis, M sediminum, M segnicrescens, M shengliensis, M squalenivorans, M similis, M
szutsaonensis, M vinifirmus, M xestospongiae, M zhanjiangensis, or M
zhejiangensis) lactate-binding protein, a Anaeromyxobacter sp. (e.g., A. dehalogenans) lactate-binding protein, a Polymorphum sp. (e.g., P. gilvum ) lactate-binding protein, a Pseudomonas sp.
(e.g., P. aeruginosa, P. alcaligenes, P. anguilliseptica, P. argentinensis, P.
borbori, P.
citronellolis, P. flavescens, P. mendocina, P. nitroreducens, P. oleovorans, P.
pseudoalcaligenes, P. resinovorans, P. straminea, P. chlororaphis, P.
asplenii, P.
aurantiaca, P. aureofaciens, P. chlororaphis, P. corrugata, P. fragi, P.
lundensis, P.
taetrolens, P. antarctica, P. azotoformans, P. blatchfordae, P.
brassicacearum, P. brenneri, P. cedrina, P. corrugata, P. fluorescens, P. gessardii, P. libanensis, P.
mandelii, P.
marginalis, P. mediterranea, P. meridiana, P. migulae, P. mucidolens, P.
orientalis, P.
panacis, P. protegens, P. proteolytica, P. rhodesiae, P. synxantha, P.
thivervalensis, P.
tolaasii, P. veronii, P. denitrificans, P. pertucinogena, P. cremoricolorata, P. entomophila, P. fulva, P. monteilii, P. mosselii, P. oryzihabitans, P. parafulva, P.
plecoglossicida, P.
putida, P. balearica, P. luteola, P. stutzeri, P. amygdali, P. avellanae, P.
caricapapayae, P.
cichorii, P. coronafaciens, P. ficuserectae, P. helianthi, P. meliae, P.
savastanoi, P. syringae, P. tomato, or P. viridiflava) lactate-binding protein, a Rhodobacter sp.
(e.g., R. aestuarii, R.
azotoformans, R. blasticus, R. capsulatus, R. johrii, R. maris, R.
megalophilus, R. ovatus, R. sphaeroides, R. veldkampii, R. vinaykumarii, or R. viridis) lactate-binding protein, a Flexistipes sp. (e.g., F. sinusarabici) lactate-binding protein, or a Thermanaerovibrio sp.
(e.g., T. acidaminovorans or T. velox) lactate-binding protein.
The present subject matter provides a ligand-binding protein that is or is a mutant of:
a Synechocystis sp. (e.g., S. sp. PCC6803) bicarbonate-binding protein, a Thermosynechococcus sp. (e.g., T. vulcanus, T. elongatus, or T. elongatus BP-1) bicarbonate-binding protein, a Chroococcidiopsis sp. (e.g., C. thermalis, C. gigantea, C.
cubana, or C.
codiicola) bicarbonate-binding protein, a Calothrix sp. (e.g., C. aberrans, C.
adscencens, C.
aeruginea, C. africana, C. allorgei, C. australiensis, C. baileyi, C.
bharadwajae, C. borealis, C. braunii, C. breviarticulata, C. calida, C. castellii, C. capitularis, C.
cavernarum Copeland, C. charicola, C. davata, C. clavatoides, C. codicola, C. columbiana, C. compacta, C. confervicola, C. contarenii, C. coriacea, C. crustacea, C. cylindrica, C.
desertica, C.
elsteri, C. epiphytica, C. evanescens, C. estonica, C. fasciculata, C.
feldmannii, C. flahaultii, C. floccosa, C. fritschii, C. fuellebornii, C. fusca, C. fusco-violacea, C.
geilterii, C. geitonos, C. ghosei, C. gigas, C. gloeocola, C. goetzei, C. hunanica, C. inaequabilis, C. inserta, C.
javanica, C. karnatakensis, C. kawraiskyi, C. kossinskajae, C. kuntzei, C.
linearis, C. minima, C. nidulans, C. parasitica, C. parietina, C. parva, C. pilosa, C. prolifera, C. pulvinata, C.
rectangularis, C. reptans, C. rodriguezii, C. santapaui, C. scopulorum, C.
scytonemicola, C.
simplex, C. simulans, C. stagnalis, C. subantarctica, C. subsimplex, C.
tenella, C. thermalis, C. twfosa, C. viguieri, C. vivipara, C. violacea, C. weberi, C. wembaerensis, C. aeruginosa, C. aestuarii, C. antarctica, C. atricha, C. bossei, C. brevissima, C. clausa, C. conica, C.
dnieprensis, C. elenkinii, C. fonticola, C. galpinii, C. gelatinosa, C.
gracilis, C. intricata, C.
litoralis, C. marchica, C. nodulosa, C. obtusa, C. rhizosoleniae, or C.
schweickertii) bicarbonate-binding protein, a Anabaena sp. (e.g., A. aequalis, A. affinis, A.
angstumalis angstumalis, A. angstumalis marchita, A. aphanizomendoides, A. azollae, A.
bornetiana, A.
catenula, A. cedrorum, A. circinalis , A. confervoides, A. constricta, A.
cyanobacterium, A.
cycadeae, A. cylindrica, A. echinispora, A. felisii, A. flos-aquae flos-aquae, A. flos-aquae minor, A. flos-aquae treleasei, A. helicoidea, A. inaequalis, A. lapponica, A.
laza, A.
lemmermannii, A. levanderi, A. limnetica, A. macrospora macrospora, A.
macrospora robusta, A. monticulosa, A. nostoc, A. oscillarioides, A. planctonica, A.
raciborskii, A.
scheremetievi, A. sphaerica, A. spiroides crassa, A. spiroides spiroides, A.
subcylindrica, A.
torulosa, A. unispora, A. variabilis, A. verrucosa, A. viguieri, A.
wisconsinense, or A.
zierlingii) bicarbonate-binding protein, or a Chamaesiphon sp. (e.g., C.
africanus, C.

amethystinus, C. britannicus, C. carpaticus, C. confervicola, C. cylindricus, C.
cylindrosporus, C. halophilus, C. incrustans, C. investiens, C. jaoi, C.
komarekii, C. longus, C. macer, C. major, C. minimus, C. minutus, C. portoricensis, C. rostafinskii, C. sideriphilus, C. tibeticus, C. aggregatus, C. fallax, C. fuscus, C. geitleri, C. mollis, C.
niger, C.
ocobyrsiodes, C. polonicus, C. polymorphus, C. starmachii, C. stratosus, or C.
subglobosus) bicarbonate-binding protein.
The present subject matter provides a ligand-binding protein that is or is a mutant of:
a Mannheimia sp. (e.g., M caviae, M glucosida, M granulomatis, M haemolytica, M
ruminalis, or M varigena) bicarbonate and iron binding protein, an Exiguobacterium sp.
(e.g., E. acetylicum, E. aestuarii, E. alkaliphilum, E. antarcticum, E.
aquaticum, E. artemiae, E. aurantiacum, E. enclense, E. indicum, E. marinum, E. mexicanum, E.
oxidotolerans, E.
profundum, E. sibiricum, E. soli, or E. undae) bicarbonate and iron binding protein, a Thermosynechococcus sp. (e.g., T. vulcanus, T. elongatus, or T. elongatus BP-1) bicarbonate and iron binding protein, a Candidatus Nitrospira sp. (e.g., Candidatus Nitrospira defluvii, Candidatus Nitrospira nitrificans, Candidatus Nitrospira nitrosa, Candidatus Nitrospira inopinata, Candidatus Magnetobacterium casensis, Candidatus Magnetobacterium bavaricum, Candidatus Magnetoovum chiemensis) bicarbonate and iron binding protein, a Thermus sp. (e.g., T. caldophilus, T. eggertssonii, T. kawarayensis, T.
murrieta, T.
nonproteolyticus, T. parvatiensis, T. rehai, T. yunnanensis, T.
amyloliquefaciens, T.
antranikianii, T. aquaticus, T. arciformis, T. brockianus, T. caliditerrae, T.
chliarophilus, T.
composti, T. filiformis, T. igniterrae, T. islandicus, T. oshimai, T.
profundus, T. scotoductus, T. tengchongensis, or T. thermophilus) bicarbonate and iron binding protein, a Meiothermus sp. (M chiliarophilus, M cerbereus, M granaticius, M rosaceus, M ruber, M
rufus, M
silvanus, M taiwanensis, or M timidus) bicarbonate and iron binding protein, a Salinibacter sp. (e.g., S. ruber, S. iranicus, or S. luteus) bicarbonate and iron binding protein, or a Halorubrum sp. (e.g., H. aidingense, H. alkaliphilum, H. arcis, H.
californiensis, H. coriense, H. distributum, H. ejinorense, H. ezzemoulense, H. kocurii, H. lacusprofundi, H. lipolyticum, H. litoreum, H. luteum, H. orientalis, H. saccharovorum, H. salsolis, H.
sodomense, H.
tebenquichense, H. terrestre, H. tibetense, H. trapanicum, H. vacuolatum, or H. xinjiangense) bicarbonate and iron binding protein.
With regard to a defined polypeptide, % identity figures higher or lower than those provided herein will encompass various embodiments. Thus, where applicable, in light of a minimum % identity figure, a polypeptide may comprise an amino acid sequence which is at least 60%, 65%, 70%, 75%, 76%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%
identical to the reference SEQ ID NO or to each of the reference SEQ ID NOs. Where applicable, in light of a maximum % identity to a reference sequence, a polypeptide may comprise an amino acid sequence which is less than 75%, 70%, 65%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, or 15% identical to the reference SEQ ID NO
or to each of the reference SEQ ID NOs. In certain embodiments, a polypeptide comprises amino acids in a sequence that is preferably at least about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% and less than about 75%, 70%, 65%, 60%, 55%, 50%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, or 30% identical to the reference SEQ ID NO
or to each of the reference SEQ ID NOs. Non-limiting examples of reference proteins and amino acid sequences disclosed herein include:
(i) a glucose-galactose binding protein from Escherichia coli (ecGGBP;
genome, NC 002695; protein, WP 032329053, SEQ ID NO: 87);
(ii) a glucose-galactose binding protein from Thermoanaerobacterium thermosaccharolyticum (ttGGBP; genome, NC_014410; protein, YP 003852930.1, SEQ ID NO: 88);
(iii) a glucose-galactose binding protein from Salmonella typhimurium (stGGBP;

genome, NC_003197; protein, WP_001036943, SEQ ID NO: 89);
(iv) a glucose-galactose binding protein from Caldicellulosiruptor hydrothermalis (chyGGBP; genome, NC_014652; protein identifier, YP_003991244.1, SEQ
ID NO: 90);
(v) a glucose-galactose binding protein from Caldicellulosiruptor obsidiansis (cobGGBP; genome, NC_014392; protein, YP_003839461.1, SEQ ID NO:
91);
(vi) a glucose-galactose binding protein from Paenibacillus sp. (pspGGBP;
genome, NC_013406; protein, YP_003243743.1, SEQ ID NO: 92);
(vii) a glucose-galactose binding protein from Clostridium saccharolyticum (csaGGBP; genome, NC_014376; protein, YP_003822565.1, SEQ ID NO:
93);

(viii) a glucose-galactose binding protein from Butyrivibrio proteoclasticus (bprGGBP; genome, NC_014387; protein, YP_003830205.1, SEQ ID NO:
94);
(ix) a glucose-galactose binding protein from Roseburia intestinalis (rinGGBP_A;
genome, NC_021012; protein, YP_007778116.1, SEQ ID NO: 95);
(x) a glucose-galactose binding protein from Faecalibacterium prausnitzii (fprGGBP; genome, NC_021020; protein, YP_007799070.1, SEQ ID NO:
96);
(xi) a glucose-galactose binding protein from Clostridium ljungdahlii (cljGGBP;
genome, NC_014328; protein, CLJU_c08950, SEQ ID NO: 97);
(xii) a glucose-galactose binding protein from Clostridium autoethanogenum (cauGGBP; genome, NC_022592; protein, CAETHG_2989, SEQ ID NO: 98);
(xiii) a glucose-galactose binding protein from Roseburia intestinalis (rinGGBP_B;
genome, NC_021012; protein, YP_007778124.1, SEQ ID NO: 99);
(xiv) a glucose-galactose binding protein from Erysipelothrix rhusiopathiae (erhGGBP; genome, NC_015601; protein, YP_004561181.1, SEQ ID NO:
100);
(xv) a glucose-galactose binding protein from Eubacterium rectale (ereGGBP;
genome, NC_012781; protein, YP_002936409.1, SEQ ID NO: 101);
(xvi) a urea-binding protein from Marinomas posidonica (mpUBP; genome, NC 015559, protein, YP 004483096.1; SEQ ID NO: 102);
(xvii) a urea-binding protein from Marinobacter hydrocarbanoclasticus (mhUBP;
genome, NC_017067, protein, YP_005430828.1; SEQ ID NO: 103);
(xviii) a urea-binding protein from Bacillus sp. (bsUBP; genome, NC_017743, protein, YP_006233530.1; SEQ ID NO: 104);
(xix) a urea-binding protein from Desulfotomaculum carboxydivorans (dsUBP;
genome, NC_015565, protein, YP_004496535.1; SEQ ID NO: 105);
(xx) a urea-binding protein from Geobacillus thermoglucosidasius (gtUBP;
genome, NC_015660, protein, YP_004588319.1; SEQ ID NO: 106);
(xxi) a urea-binding protein from Clostridium thermocellum (ctUBP; genome, NC 009012, protein, YP 001038237.1; SEQ ID NO: 107);
(xxii) a urea-binding protein from Caldicellulosiruptor saccharolyticus (csUBP;
genome, NC_009437, protein, YP_001181243.1; SEQ ID NO: 108);

(xxiii) a urea-binding protein from Thermocrinis albus (taUBP; genome, NC 013894, protein, YP 003473480.1; SEQ ID NO: 109);
(xxiv) a urea-binding protein from Geobacillus kaustophilus (gkUBP; genome, NC 006510, protein, YP 147790.1; SEQ ID NO: 110);
(xxv) a urea-binding protein from Paenibacillus sp. (psUBP; genome, NC_013406, protein, YP_003241723.1; SEQ ID NO: 111);
(xxvi) a urea-binding protein from Thermosynechococcus elongatus (teUBP;
genome, NC_004113, protein, YP_681910.1; SEQ ID NO: 112);
(xxvii) a glucose-binding protein from Thermus thermophilus (ttGBP; genome, NC 005835, protein, YP 004303.1 and WP 011172778; SEQ ID NO: 113);
(xxviii)a glucose-binding protein from Thermus scotoductus (tsGBP; genome, NC 014974, protein, YP 004202647.1; SEQ ID NO: 114);
(xxix) a glucose-binding protein from Deinococcus maricopensis (dmGBP; genome, NC 014958, protein, YP 004171760.1; SEQ ID NO: 115);
(xxx) a glucose-binding protein from Thermotoga neapolitana (tnGBP; genome, NC 011978, protein, YP 002534202.1; SEQ ID NO: 116);
(xxxi) a glucose-binding protein from Kosmotoga olearia (koGBP; genome, NC 012785, protein, YP 0029416871.1; SEQ ID NO: 117);
(xxxii) a glucose-binding protein from Bacillus halodurans (bhGBP; genome, NC 002570, protein, YP 244712.1; SEQ ID NO: 118);
(xxxiii)a glucose-binding protein from Staphylothermus marinus (smGBP; genome, NC 009033, protein, YP 001041152.1; SEQ ID NO: 119);
(xxxiv)a glucose-binding protein from Arthrobacter sp. (asGBP; genome, NC 008541, protein, YP 8313491.1; SEQ ID NO: 120);
(xxxv) a lactate-binding protein from Thermus thermophilus (ttLacBP; genome, NC 006461, protein, YP 144032.1; SEQ ID NO: 121);
(xxxvi)a lactate-binding protein from Thermus scotoductus (tscLacBP; genome, NC 014974, protein YP 004202714.1; SEQ ID NO: 122);
(xxxvii) a lactate-binding protein from Thermus oshimai (toLacBP; genome, NC 019386, protein YP 019386; SEQ ID NO: 123);
(xxxviii) a lactate-binding protein from Thioalkalivibrio sulfidophilus (tsuLacBP; genome, NC_011901, protein YP_002514099.1; SEQ ID NO:
124);

(xxxix)a lactate-binding protein from Roseobacter denitrificans (rdLacBP;
genome, NC 008209, protein YP 683924.1; SEQ ID NO: 125);
(xl) a lactate-binding protein from Marinobacter sp. (msLacBP; genome, NC 018268, protein YP 006556686.1; SEQ ID NO: 126);
(xli) a lactate-binding protein from Thermus sp. (tspLacBP; genome, NC_017278, protein YP_005654632.1; SEQ ID NO: 127);
(xlii) a lactate-binding protein from Marinobacter adhaerens (maLacBP; genome, NC 017506, protein YP 005686720.1; SEQ ID NO: 128);
(xliii) a lactate-binding protein from Anaeromyxobacter dehalogens (adLacBP;
genome, NC_007760, protein YP_466_099.1; SEQ ID NO: 129);
(xliv) a lactate-binding protein from Polymorphum gilvum (pgLacBP; genome, NC 015259, protein YP 4304976.1; SEQ ID NO: 130);
(xlv) a lactate-binding protein from Pseudomonas stuztzeri (psLacBP; genome, NC 018177, protein YP 00652676.1; SEQ ID NO: 131);
(xlvi) a lactate-binding protein from Rhodobacter sphaeroides (rsLacBP;
genome, NC 007494, protein RSP 3372; SEQ ID NO: 132);
(xlvii) a lactate-binding protein from Flexistipes sinusarabici (fsLacBP;
genome, NC 015672, protein YP 004603455.1; SEQ ID NO: 133);
(xlviii) a lactate-binding protein from Thermanaerovibrio acidaminovorans (taLacBP;
genome, NC_013522, protein YP_003317968.1; SEQ ID NO: 134);
(xlix) a bicarbonate-binding protein from Synechocystis sp. (synBicarbBP1;
genome, NC 017052, protein YP 005410477.1; SEQ ID NO: 135);
(1) a bicarbonate-binding protein from Thermosynechococcus elongatus (teBicarbBP2; genome, NC_004113, protein NP_682790.1; SEQ ID NO:
136);
(1i) a bicarbonate-binding protein from Chroococcidiopsis thermalis (ctBicarbBP3; genome, NC_019695, protein YP_007090308.1; SEQ ID NO:
137);
(lii) a bicarbonate-binding protein from Calothrix sp. (calBicarbBP4; genome, NC 019751, protein YP 007137061.1; SEQ ID NO: 138);
(liii) a bicarbonate-binding protein from Anabaena variabilis (avBicarbBP5;
genome, NC_007413, protein YP_321546.1; SEQ ID NO: 139);

(liv) a bicarbonate-binding protein from Chamaesiphon minutus (cmBicarbBP6;
genome, NC_019697, protein YP_007099445.1; SEQ ID NO: 140);
(1v) a bicarbonate and iron binding protein from Mannheimia haemolytica (mhFeBP1; genome, NC_0121082, protein, YP_007884192.1; SEQ ID NO:
141);
(lvi) a bicarbonate and iron binding protein from Exiguobacterium sp.
(exiFeBP2;
genome, NC_012673, protein, YP_002886303.1; SEQ ID NO: 142);
(lvii) a bicarbonate and iron binding protein from Thermosynechoccus elongatus (teFeBP3; genome, NC_004113, protein, NP_681303.1; SEQ ID NO: 143);
(lviii) a bicarbonate and iron binding protein from Candidatus nitrospira (cnFeBP4;
genome, NC_014355, protein, YP_003796723.1; SEQ ID NO: 144);
(lix) a bicarbonate and iron binding protein from Thermus thermophilus (ttFeBP5;
genome, NC_006461, protein, YP_144894.1; SEQ ID NO: 145);
(1x) a bicarbonate and iron binding protein from Meiothermus silvanus (msFeBP6;
genome, NC_014212, protein, YP_003686074.1; SEQ ID NO: 146);
(lxi) a bicarbonate and iron binding protein from Salinibacter ruber (srFeBP7;

genome, NC_014032, protein, YP_003572493.1; SEQ ID NO: 147); and (lxii) a bicarbonate and iron binding protein from Halorubrum lacusprofundi (h1FeBP8; genome, NC_012029, protein, YP_002564837.1; SEQ ID NO:
148).
The ligand-binding proteins disclosed herein may optionally be fused (e.g., at their N-terminal and/or C-terminal ends) to a motif comprising a stretch of amino acids that facilitates the isolation or other manipulation such as conjugation to a moiety or immobilization on a substrate such as a plastic, a cellulose product such as paper, polymer, metal, noble metal, semi-conductor, or quantum dot (e.g., a fluorescent quantum dot) . A
non-limiting example of such a stretch of amino acids has the sequence:
GGSHHHHHH
(SEQ ID NO: 152). This motif is not required for, is not believed to influence or affect ligand-binding activity or signal transduction, and may be omitted from any ligand-binding protein or biosensor disclosed herein. Additionally, for every sequence disclosed herein that includes GGSHHHHHH (SEQ ID NO: 152), a corresponding sequence that is identical except that it lacks GGSHHHHHH (SEQ ID NO: 152) is also provided and intended to be disclosed. For example, each of SEQ ID NOs: 1-41 (and the non-limiting examples of other proteins used in the experiments disclosed herein) comprises this motif (SEQ
ID NO: 152).

Alternatively or in addition, a ligand-binding protein may be fused to a non-native polypeptide or "added amino acids" that facilitates the attachment thereof to a surface, such as the surface of a device.
In some embodiments, a polypeptide comprises 1, 2, 3, 4, 5, or more substitutions or deletions of a cysteine compared to the naturally occurring counterpart of the polypeptide (i.e., 1, 2, 3, 4, 5, or more native cysteines have been removed), e.g., 1, 2, 3, 4, 5, or more cysteine to alanine substitutions compared to the naturally occurring counterpart of the polypeptide. In some embodiments, all of the cysteines of a polypeptide have been deleted and/or substituted compared to its natural counterpart. In some embodiments, one or more cysteines of a polypeptide have been substituted with an alanine, a serine, or a threonine.
In embodiments, the amino acid sequence of a protein comprises no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 mutations compared to its naturally occurring counterpart. In some embodiments, less than 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, or 2 of the mutations is a deletion or insertion of 1, 2, 3, 4, or 5 or no more than 1, 2, 3, 4, or 5 amino acids. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more of the mutations is a substitution mutation. In certain embodiments, every mutation to a protein compared to its naturally occurring counterpart is a substitution mutation. In various embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more or all of the mutations to a protein compared to its naturally occurring counterpart is a conservative substitution mutation.
In various embodiments, a polypeptide does not have any insertion or deletion compared to its natural counterpart, other than (optionally) the removal of the signal peptide and/or the fusion of compounds such as another polypeptide at the N-terminus or C-terminus thereof.
Exemplary Ligand-Binding Proteins Various biosensors provided herein comprise ligand-binding proteins, such as proteins that have altered amino acid sequences compared to their naturally occurring counterparts. In embodiments, such proteins are conjugated to reporter groups.
In various embodiments, the Ca, root-mean-square deviation (RMSD) between the backbone of a ligand-binding protein and its naturally occurring counterpart is, e.g., between about 0-3 A, 0-1 A, 0-1.5 A, 0-2 A, 0.1-3 A, 0.5-1 A, 0.5-1.5 A, or 0.5-2 A, or less than about 0.1 A, 0.2 A, 0.3 A, 0.4 A, 0.5 A, 0.6 A, 0.7 A, 0.8 A, 0.9 A, 1.0 A, 1.5 A, 1.6 A, 1.7 A, 1.8 A, 1.9 A, 2.0 A, 2.5 A, or 3 A. In some embodiments, the Ca RMSD between the N-terminal domain (i.e., the portion of the protein at the N-terminal side of the binding domain hinge) backbone of the ligand-binding protein and the corresponding domain of its naturally occurring counterpart is, e.g., between about 0-3 A, 0-1 A, 0-1.5 A, 0-2 A, 0.1-3 A, 0.5-1 A, 0.5-1.5 ik, or 0.5-2 ik, or less than about 0.1 ik, 0.2 ik, 0.3 ik, 0.4 ik, 0.5 ik, 0.6 ik, 0.7 ik, 0.8 A, 0.9 A, 1.0 A, 1.5 A, 1.6A, 1.7A, 1.8A, 1.9 A, 2.0 A, 2.5 A, or 3 A. In certain embodiments, the Ca RMSD between the C-terminal domain (i.e., the portion of the protein at the C-terminal side of the binding domain hinge) backbone of the ligand-binding polypeptide and the corresponding domain of its naturally occurring counterpart is, e.g., between about 0-3 A, 0-1 A, 0-1.5 A, 0-2 A, 0.1-3 A, 0.5-1 A, 0.5-1.5 A, or 0.5-2 A, or less than about 0.1 ik, 0.2 ik, 0.3 ik, 0.4 ik, 0.5 ik, 0.6 ik, 0.7 ik, 0.8 ik, 0.9 ik, 1.0 ik, 1.5 ik, 1.6 ik, 1.7 A, 1.8 A, 1.9 A, 2.0 A, 2.5 A, or 3 A. Non-limiting considerations relating to the sequence and structural differences between homologous proteins are discussed in Chothia and Lesk (1986) The EMBO Journal, 5(4):823-826, the entire content of which is incorporated herein by reference.
Non-limiting examples of ligand-binding polypeptides that are useful in biosensors provided herein include variants of the naturally occurring proteins disclosed herein that comprise cysteine substitutions and/or N-terminal and/or C-terminal fusions (e.g., to a fluorophore attachment moiety). In embodiments, a biosensor comprises a modified ligand-binding protein having an amino acid substitution compared to its naturally occurring counterpart, such that the polypeptide has a cysteine at one or more of position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, or 400 or any combination of 1, 2, 3, 4, or 5 thereof, wherein the position corresponds a SEQ ID NO disclosed herein. In embodiments, the cysteine is conjugated to a reporter group.
In various embodiments, the dissociation constant of the mutant ligand-binding polypeptide differs by at least about 1 1.1,M, 5 1.1,M, 10 ,M, 20 1.1,M, 25 ,M, 30 ,M, 35 1.1,M, 40 1.1,M, 45 1.1,M, 50 ,M, 75 ,M, 100 ,M, 200 ,M, 300 1.1,M, 400 1.1,M, 500 1.1,M, 600 ,M, 700 ,M, 800 1.1,M, 900 1.1,M, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, or 100 mM (increase or decrease) compared to its naturally occurring counterpart.
The biosensors and ligand-binding proteins provided herein are robust and useful at a wide range of physical conditions, e.g., pressure, temperature, salinity, osmolality, and pH
conditions. For example, biosensors and ligand-binding proteins provided herein may survive substantial periods of time after being dried or exposed to high temperatures. In some embodiments, the biosensor maintains at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or more of its signal transduction activity after exposure to a temperature of about 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, or 125, or 40-125 C for about 1, 2, 3, 4, 5, 6, 15, 30, 60, 120, 180, 240, or 360 minutes. In certain embodiments, the biosensor maintains at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or more of its signal transduction activity after 1, 2, 3, 4, or 5 freeze-thaw cycles in an aqueous solution. In various embodiments, the biosensor maintains at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or more of its signal transduction activity after storage at a temperature of between 20-37 C
for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, or 1-24 months in dry form. In some embodiments, the optimal functional temperature of the biosensor is between 41 and 122 C, between 20 and 40 C, or less than about 10 C (e.g., between -20 and +10 C).
Devices, compositions, and biosensors provided herein may be stored, e.g., with or without protection from exposure to light. In some embodiments, the devices, compositions, and biosensors are stored in the dark, e.g., with protection from light.
Non-limiting examples of glucose-binding proteins include variants of ecGGBP, ttGGBP, stGGBP, chyGGBP, cobGGBP, pspGGBP, csaGGBP, bprGGBP, rinGGBP_A, rinGGBP_B, fprGGBP, cljGGBP, cauGGBP, erhGGBP, ereGGBP, and chyGGBP.
In embodiments, a biosensor comprises a modified ecGGBP. In non-limiting examples, the modified ecGGBP may comprise one or more, or any combination of the following substitutions compared to its naturally occurring counterpart: Y10X, D14X, N15X, F16X, P7OX, N91X, K92X, S112X, 5115X, E149X, H152X, P153X, D154X, A155X, R158X, M182X, W183X, N211X, D212X, D236X, L238X, L255X, N256X, D257X, P294X, and V296X, where X is any amino acid, an amino acid that results in a conservative substitution, or a cysteine, and where each position is counted in ecGGBP
without including the signal peptide (SEQ ID NO: 153). In some embodiments, the modified ecGGBP
comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 of the following substitutions:
YlOA, YlOC, D14C, D14A, D14Q, D14N, D145, D14T, D14E, D14H, D14L, D14Y, D14F, N15C, F16L, F16A, F16C, F16Y, N91C, N91A, K92A, K92C, E93C, S112A, S115A, E149C, E149K, E149Q, E1495, H152C, H152A, H152F, H152Q, H152N, D154C, D154A, D154N, A155C, A1555, A155H, A155L, A155F, A155Y, A155N, A155K, A155M, A155W, A155Q, R158C, R158A, R158K, M182C, M182W, W183C, W183A, N211C, N211F, N211W, N211K, N211Q, N2115, N211H, N211M, N211C, D212C, L238C, D236C, D236A, D236N, L255C, N256A, N256D, D257C, P294C, and V293C.
In various embodiments, a biosensor comprises a modified ttGGBP. In non-limiting examples, the modified ttGGBP may comprise one or more, or any combination of the following substitutions compared to its naturally occurring counterpart: YllX, D15X, T16X, F17X, G20X, N42X, V67X, R69X, R91X, E92X, Al 11X, Q148X, H151X, Q152X, A154X, N181X, W182X, D183X, D211X, T237X, T240X, L257X, N258X, D259X, A260X, and K300X where X is any amino acid, an amino acid that results in a conservative substitution, or a cysteine, and where each position is counted in ttGGBP with the signal peptide replaced with a methionine (SEQ ID NO: 154). In some embodiments, the modified ttGGBP
comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 of the following substitutions:
Y11C, D15A, D15E, D15N, DISC, T165, T16N, T16C, F17C, G20A, G20C, N42C, V67C, R69P, R69C, R91K, E92C, AMC, Q1485, Q148K, Q148E, Q148C, H151Q, H151N, H151F, H151C, Q152P, Q152C, A1545, A154N, A154M, A154F, A154C, N181C, W182C, D183C, D211A, D211C, T237C, T240A, L257C, N258D, N258S, N258A, N258C, D259C, A260N, A260Q, A260R, A260K, A260W, A260F, A260Y, A260S, A260C, and K300C.
In embodiments, a biosensor comprises a modified stGGBP. In non-limiting examples, the modified stGGBP may comprise one or more, or any combination of the following substitutions compared to its naturally occurring counterpart: Y1 1X, Y13X, D15X, N16X, F17X, P71X, N92X, K93X, P113X, S116X, E150X, H153X, P154X, D155X, A156X, R159X, M183X, W184X, N211X, N212X, D213X, A214X, D237X, L239X, D258X, P295X, and V297X where X is any amino acid, an amino acid that results in a conservative substitution, or a cysteine, and where each position is counted in stGGBP with the signal peptide replaced with a methionine (SEQ ID NO: 155). In some embodiments, the modified stGGBP comprises 1, 2 or 3 of the following mutations: Y13C, F17C, and W184C.
In embodiments, a biosensor comprises a modified chyGGBP. In non-limiting examples, the modified chyGGBP may comprise one or more, or any combination of the following substitutions compared to its naturally occurring counterpart: F12X, D14X, T15X, F16X, R68X, N89X, R90X, All0X, 5113X, E147X, H150X, Q151X, D152X, A153X, R156X, M180X, W181X, N207X, N208X, D209X, D210X, D237X, T239X, D258X, V296X, and Y298X where X is any amino acid, an amino acid that results in a conservative substitution, or a cysteine, and where each position is counted in chyGGBP
with the signal peptide replaced with a methionine (SEQ ID NO: 156). In some embodiments, the modified chyGGBP comprises 1, 2, or 3 of the following mutations: F12C, F16C, C39A, W181C, and C206A.
In embodiments, a biosensor comprises a modified cobGGBP. In non-limiting examples, the modified cobGGBP may comprise one or more, or any combination of the following substitutions compared to its naturally occurring counterpart: F12X, D14X, T15X, F16X, C39X, R68X, N89X, R90X, Al 10X, 5113X, E147X, H150X, Q151X, D152X, A153X, R156X, C173X, M180X, W181X, C206X, N207X, N208X, D209X, D210X, D237X, T239X, D258X, P297X, and Q299X where X is any amino acid, an amino acid that results in a conservative substitution, or a cysteine, and where each position is counted in cobGGBP with the signal peptide replaced with a methionine (SEQ ID NO: 157).
In some embodiments, the modified cobGGBP comprises 1, 2, or 3 of the following mutations: Fl 2C, F16C, C39A, C173A, W181C, and C206A.
In embodiments, a biosensor comprises a modified pspGGBP. In non-limiting examples, the modified pspGGBP may comprise one or more, or any combination of the following substitutions compared to its naturally occurring counterpart: F9X, D11X, T12X, F13X, R65X, N86X, R87X, A107X, 5110X, E144X, H147X, Q148X, D149X, A150X, R153X, M177X, W178X, N204X, N205X, D206X, D207X, D234X, T236X, 255X, A294X, and K296X where X is any amino acid, an amino acid that results in a conservative substitution, or a cysteine, and where each position is counted in pspGGBP
with the signal peptide replaced with a methionine (SEQ ID NO: 158). In some embodiments, the modified pspGGBP comprises 1, 2, or 3 of the following mutations: F9C, F13C, and W178C.
In embodiments, a biosensor comprises a modified csaGGBP. In non-limiting examples, the modified csaGGBP may comprise one or more, or any combination of the following substitutions compared to its naturally occurring counterpart: Y14X, D16X, F18X, C62X, I72X, C82X, N93X, R94X, C113A, S118X, A121X, E152X, N155X, E156X, D157X, S158X, R161X, N185X, W186X, C211X, D241X, L243X, D262X, D290X, I292X, I297X, F299X, Q301X, and T302X where X is any amino acid, an amino acid that results in a conservative substitution, or a cysteine, and where each position is counted in csaGGBP
with the signal peptide replaced with a methionine (SEQ ID NO: 159). In some embodiments, the modified csaGGBP comprises 1, 2, 3, 4, 5, 6, 7, or 8 of the following mutations: Y14C, F18C, C62A, C82A, C113A, W186C, and C211A.
In embodiments, a biosensor comprises a modified bprGGBP. In non-limiting examples, the modified bprGGBP may comprise one or more, or any combination of the following substitutions compared to its naturally occurring counterpart: C8X, K12X, D14X, N15X, F16X, 572X, N93X, R94X, C112X, C116X, Al 18X, 5121X, A153X, N156X, I157X, D158X, A159X, C179X, N186X, W187X, C211X,N212X,N213X, D214X, A215X, D241X, D243X, K251X, C289X, D290X, and V292X where X is any amino acid, an amino acid that results in a conservative substitution, or a cysteine, and where each position is counted in bprGGBP with the signal peptide replaced with a methionine (SEQ ID
NO: 160).
In some embodiments, the modified bprGGBP comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 of the following mutations: C8A, K12C, F16C, C112A, C116A, C179A, W187C, C211A, and C289A.
In embodiments, a biosensor comprises a modified rinGGBP_A. In non-limiting examples, the modified rinGGBP_A may comprise one or more, or any combination of the following substitutions compared to its naturally occurring counterpart: C6X, Fl OX, D12X, N13X, F14X, 570X, N91X, R92X, C114X, Al 16X, Q118X, D151X, N154X, V155X, D156X, A157X, R160X, C177X, N184X, W185X, C210X, N211X, N212X, D213X, A214X, D240X, L242X, L250X, C288X, D289X, and V291X where X is any amino acid, an amino acid that results in a conservative substitution, or a cysteine, and where each position is counted in rinGGBP_A with the signal peptide replaced with a methionine (SEQ ID NO:
161). In some embodiments, the modified rinGGBP_A comprises 1, 2, 3, 4, 5, 6, 7, or 8 of the following mutations: C6A, FlOC, F14C, C114A, C177A, W185C, C210A, and C288A.
In embodiments, a biosensor comprises a modified rinGGBP_B. In non-limiting examples, the modified rinGGBP_B may comprise one or more, or any combination of the following substitutions compared to its naturally occurring counterpart: Q13X, D15X, T16X, F17X, C66X, C70A, R76X, N97X, R98X, Al 18X, S121X, E155X, H158X, Q159X, D160X, A161X, R164X, N188X, W189X, N215X,N216X, D217X, D218X, D244X, T246X, D265X, P301X, A303X, and C306X where X is any amino acid, an amino acid that results in a conservative substitution, or a cysteine, and where each position is counted in rinGGBP_B
with the signal peptide replaced with a methionine (SEQ ID NO: 165). In some embodiments, the modified rinGGBP_B comprises 1, 2, 3, 4, 5, or 6 of the following mutations: Q13C, F17C, C66A, C70A, W189C, and C306A.
In embodiments, a biosensor comprises a modified fprGGBP. In non-limiting examples, the modified fprGGBP may comprise one or more, or any combination of the following substitutions compared to its naturally occurring counterpart: C8A, Fl 2X, D14X, N15X, F16X, T69X, N90X, R91X, C105X, C106X, Al 13X, 5116X, C143X, D146X, N149X, 1150X, D151X, A152X, R155X, N179X, W180X, C205A, N206X, N207X, D208X, A209X, D235X, L237X, N243X, D284X, and V286X where X is any amino acid, an amino acid that results in a conservative substitution, or a cysteine, and where each position is counted in fprGGBP with the signal peptide replaced with a methionine (SEQ ID
NO: 162).
In some embodiments, the modified fprGGBP comprises 1, 2, 3, 4, 5, 6, or 7 of the following mutations: C8A, F12C, F16C, C105A, C106A, C143A, W180C, and C205A.
In embodiments, a biosensor comprises a modified cljGGBP. In non-limiting examples, the modified cljGGBP may comprise one or more, or any combination of the following substitutions compared to its naturally occurring counterpart: F11X, N13X, T14X, W15X, V67X, C77X, N88X, R89X, A109X, S112X, E142X, N145X, Q146X, D147X, A148X, R151X, M175X, W176X, C198X, N201X, N202X, D203X, D204X, D231X, T233X, D252X, D291X, and K294X where X is any amino acid, an amino acid that results in a conservative substitution, or a cysteine, and where each position is counted in cljGGBP
with the signal peptide replaced with a methionine (SEQ ID NO: 163). In some embodiments, the modified cljGGBP comprises 1, 2, 3, 4, or 5 of the following mutations:
Fl1C, W15C, C77A, W176C, and C198A.
In embodiments, a biosensor comprises a modified cauGGBP. In non-limiting examples, the modified cauGGBP may comprise one or more, or any combination of the following substitutions compared to its naturally occurring counterpart: F12X, N14X, T15X, W16X, V68X, C78X, N89X, R90X, Al 10X, S113X, E143X, N146X, Q147X, D148X, A149X, R152X, M176X, W177X, C199X, N203X, N204X, D205X, D206X, D233X, T235X, D254X, D293X, and K295X where X is any amino acid, an amino acid that results in a conservative substitution, or a cysteine, and where each position is counted in cauGGBP
with the signal peptide replaced with a methionine (SEQ ID NO: 164). In some embodiments, the modified cauGGBP comprises 1, 2, 3, 4, or 5 of the following mutations:
F12C, W16C, C78A, W177C, and C199A.
In embodiments, a biosensor comprises a modified erhGGBP. In non-limiting examples, the modified erhGGBP may comprise one or more, or any combination of the following substitutions compared to its naturally occurring counterpart: F13X, D15X, N16X, F17X, P76X, N97X, R98X, Al 19X, S122X, D153X, N156X, V157X, D158X, A159X, R162X, N187X, W188X, N214X, N215X, D216X, G217X, D243X, I245X, D264X, E312X, and V314X where X is any amino acid, an amino acid that results in a conservative substitution, or a cysteine, and where each position is counted in erhGGBP
with the signal peptide replaced with a methionine (SEQ ID NO: 166). In some embodiments, the modified erhGGBP comprises 1, 2, or 3 of the following mutations: F13C, F17C, and W188C.
In embodiments, a biosensor comprises a modified ereGGBP. In non-limiting examples, the modified ereGGBP may comprise one or more, or any combination of the following substitutions compared to its naturally occurring counterpart: Q13X, D15X, T16X, F17X, C29X, C65X, C69X, R75X, N96X, R97, Al 17X, 5120X, E154X, H157X, Q158X, D159X, A160X, R163X, C183X, N187X, W188X,N214X, N215X, D216X, A217X, D243X, T245X, D264X, P301X, and E303X where X is any amino acid, an amino acid that results in a conservative substitution, or a cysteine, and where each position is counted in ereGGBP with the signal peptide replaced with a methionine (SEQ ID NO: 167).
In some embodiments, the modified ereGGBP comprises 1, 2, 3, 4, 5, 6, 7, or 8 of the following mutations: ClOA, Q13C, F17C, C29A, C65A, C69A, C183A, and W188C.

Fluorescent Proteins Various biosensors provided herein comprise fluorescent proteins, such as fluorescent proteins that have altered amino acid sequences compared to their naturally occurring counterparts. In embodiments, such proteins are conjugated to reporter groups.
In embodiments, the proteins are not conjugated to a reporter group (i.e., a biosensor comprising the fluorescent protein that does not undergo tgmFRET or ngmFRET is provided).
Aspects of the present subject matter provide a biosensor for ligand, comprising a ligand-binding protein, wherein the ligand-binding protein is a fluorescent protein, and wherein binding of the ligand to a ligand-binding domain of the fluorescent protein causes a change in fluorescence by the fluorescent protein. In some embodiments, the biosensor further comprises a reporter group, e.g., a fluorophore that acts as a ngmFRET
donor fluorophore or a ngmFRET acceptor fluorophore with respect to the fluorescent protein.
Green Fluorescent Protein (GFP) and its derivatives such as Yellow Fluorescent Protein (YFP) form their internal fluorophore through an autocatalytic, posttranslational cyclization of a tipeptide from its own amino acid sequence (M. Zimmer, 2002, Chem. Rev.
102, 759-781). This process entails three steps: a nucleophilic attack to create a cyclic peptide, dehydration, and a final oxidation to introduce conjugation (D.P.
Barondeau et al., 2003, Proc. Natl. Acad. Sci. USA, 100, 12111-12116). The formation of GFP's or YFP's fluorophore is an autocatalytic process that requires no catalyst external to these proteins.
In some embodiments, the ligand comprises a halide anion such as a fluoride (F), chloride (Cr), a bromide (BC), an iodide (n, astatide (At-) anion, or an ununseptide (Ts-) anion. In certain embodiments, the fluorescent protein has an affinity (Kd) for the halide anion that is within the concentration range of the halide anion in a subject.
Non-limiting examples of fluorescent proteins that bind halide anions include Yellow Fluorescent Protein (YFP; SEQ ID NO: 149) and mutants thereof. In some embodiments, the fluorescent protein comprises a mutation that alters the interaction of the mutant with a bound halide anion compared to YFP. For example, the mutation that may alter the fluorescent protein's affinity and/or specificity for a halide anion compared to YFP. In various embodiments, the fluorescent protein comprises 1 halide anion binding site. Alternatively the fluorescent protein comprises at least 2, 3, 4, or 5 halide anion binding sites. In some embodiments, at least one amino acid of the YFP has been substituted with a cysteine.
YFP is a non-limiting reference protein respect to fluorescence proteins.
In various embodiments, a polypeptide of the present disclosure comprises (a) amino acids in a sequence that is preferably (i) at least about 10%, 11%, 12%, 13%, 14%, or 15%, and (ii) less than about 75%, 70%, 65%, 60%, 55%, 50%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, or 35% identical to YFP;
(b) a stretch of at least 5, 10, or 20 amino acids having at least about 50%, 55%, 60%, 65%, 75%, 80%, 85%, 90%, or 95% identity to a stretch of consecutive amino acids including position 17, 32, 43, 77, 95, 109, 122, 133, 149, 164, 173, 182, 204, and/or 221 of YFP;
(c) no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 15 deleted or inserted amino acids compared to YFP, not including added amino acids added to the N-terminus or C-terminus of the polypeptide compared to its natural counterpart, and including or not including the signal peptide of the natural counterpart of the polypeptide;
(d) at least 9, 10, 11, or 12 f3-strands or exactly 9, 10, 11, or 12 f3-strands.
In some embodiments, the fluorescent polypeptide comprises a f3-barrel. In certain embodiments, the f3-barrel comprises 9, 10, 11, or 12 f3-strands. In some embodiments, the fluorescent protein comprises a cysteine within the first f3-strand (f31), the second f3-strand (132), the third f3-strand (f33), the fourth f3-strand (f34), the fifth f3-strand (f35), the sixth f3-strand (f36), the seventh f3-strand (f37), the eighth f3-strand (f38), the ninth f3-strand (f39), the tenth f3-strand (f310), or the eleventh f3-strand (f3n) of a YFP. In some embodiments, the polypeptide comprises (i) 1, 2, or 3 amino acid substitutions between f31 and f32; (ii) 1, 2, or 3 amino acid substitutions between f32 and f33; (iii) 1, 2, or 3 amino acid substitutions between the f33 and J34; (iv) 1, 2, or 3 amino acid substitutions between the f34 and f35; (v) 1, 2, or 3 amino acid substitutions between f35 and f36; (vi) 1, 2, or 3 amino acid substitutions between f36 and f37;
(vii) 1, 2, or 3 amino acid substitutions between the f37 and f38; (viii) 1, 2, or 3, amino acid substitutions between f38 and f39; (ix) 1, 2, or 3 amino acid substitutions between the f39 and f310; and/or (x) 1, 2, or 3 amino acid substitutions between f310 and f311. In various embodiments, the 1 or more substitutions comprise a substitution with cysteine. In certain embodiments, the cysteine follows f3i 1 in the amino acid sequence of the YFP.
Alpha-helical and f3-strand segments assignments are calculated from a three-dimensional protein structure as follows, and as described in C.A.F. Andersen, B. Rost, 2003, Structural Bioinformatics, 341-363, P.E. Bourne, ed., Wiley, the entire content of which is incorporated herein by reference. First for a given residue, i, the backbone trace angle, r, is calculated, defined as the dihedral angle between the four successive Ca, atom positions of residues in the linear protein sequence i, i+1, i+2, i+3. These values are calculated for all residues. Second, the residues that form backbone hydrogen bonds with each other are recorded. A hydrogen bond is scored if the distance between the backbone amide nitrogen and carbonyl oxygen of two different residues in the protein is calculated to be 2.5A or less, and if the calculated angle between the nitrogen, its amide proton, and the carbonyl is greater than 120 . A residue is deemed to be in an a-helix, if 35 r 65, and it makes a backbone hydrogen bond with its i+4th neighbor in the linear amino acid sequence. It is deemed to be in a (3-strand, if the absolute t value falls in the interval 120 k-1 180 and if it makes at least one hydrogen bond with another residue with the same r value range. Alpha-helical segments comprise at least four residues; (3-strand residues comprise at least three residues.
In embodiments, a biosensor comprises a modified YFP polypeptide having an amino acid substitution compared to its naturally occurring counterpart, such that the polypeptide has a cysteine at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,

16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, or 250, or any combination of 1, 2, 3, 4, or 5 thereof, wherein the position corresponds a SEQ ID NO
disclosed herein for YFP. In embodiments, the cysteine is conjugated to a reporter group.
In embodiments, a biosensor comprises a modified YFP. In non-limiting examples, the modified YFP may comprise one or more, or any combination of the following substitutions compared to its naturally occurring counterpart: E17X, E32X, T43X, F64X, G65X, L68X, Q69X, A72X, H77X, K79X, R80X, E95X, R109X, R122X, D133X, H148X, N149X, V163X,N164X, D173X, Y182X, Q183X, Y203X, Q204X, L221X, and H231X, where X is any amino acid or is an amino acid that results in a conservative substitution. In some embodiments X is cysteine. In some embodiments, the modified YFP
comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 of the following substitutions: F64L, G65T, L68V, Q69T, A72S, K79R, R80Q, H148Q, H148G, V163A, H231L, H148Q, or Q183A, wherein each YFP
amino acid position is numbered as in SEQ ID NO: 150. In some embodiments, the fluorescent protein comprises an R at the 96 position, a Y at the 203 position, a S at the 205 position, and an E at the 222 position compared to YFP, wherein each YFP amino acid position is numbered as in SEQ ID NO: 150. C1BP1 (also referred to as laYFP) comprises L68V, K79R, R80Q, H2131L compared to YFP (as numbered in SEQ ID NO: 150).
Adaptor Proteins Aspects provide biosensor for a ligand, comprising (a) a polypeptide; (b) a directly responsive fluorophore, wherein binding of a ligand to the directly responsive fluorophore causes a change in signaling by the directly responsive fluorophore (i.e., the fluorophore is chemoresponsive); and (b) an indirectly responsive fluorophore. The directly responsive fluorophore may be a donor fluorophore or an acceptor fluorophore. In some embodiments, the directly responsive fluorophore is a donor fluorophore and the indirectly responsive fluorophore is an acceptor fluorophore. In some embodiments, the directly responsive fluorophore is an acceptor fluorophore and the indirectly responsive fluorophore is a donor fluorophore. ngmFRET occurs between the donor fluorophore and the acceptor fluorophore when the donor fluorophore is contacted with radiation comprising the excitation wavelength of the donor fluorophore.
Any polypeptide may be used to link a directly responsive fluorophore (e.g., a chemoresponsive fluorophore) with an indirectly responsive fluorophore. Such a polypeptide is referred to as an adaptor protein herein. In some embodiments, the polypeptide comprises a stretch of at least 2, 3, 4, 5, 6, 47, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, or 500 amino acids.
In some embodiments, the polypeptide comprises a stretch of at least 50, 60, 70, 80, 90, or 100 amino acids in a sequence that is at least about 85%, 90%, 95%, or 99% identical to the amino acid sequence of ecTRX (SEQ ID NO: 151). In certain embodiments, the polypeptide comprises at least 1, 2, or 3 thiol groups; at least 1, 2, or 3 cysteines that each comprise a sulfhydryl group; at least 1, 2, or 3 primary amine groups; or at least 1, 2, or 3 lysines that each comprise a primary amine. In various embodiments, there is no disulfide bond between cysteines within the amino acid sequence of the polypeptide.

In some embodiments, the polypeptide comprises a mutant of ecTRX comprising a D3X, K4X, K19X, D27X, K37X, K53X, K58X, K70X, R74X, K83X, K91X, K97X, or K101X mutation, or any combination thereof, wherein X is any amino acid, and wherein each ecTRX amino acid position is numbered as in SEQ ID NO: 151.
In various embodiments, the polypeptide comprises a mutant of ecTRX comprising a D3A, D3A, K4R, K4Q, K19R, K19Q, D27A, K37R, K53M, K53R, K58M, K7OR, R74C, K83R, K91R, K97R, or K101R mutation, or any combination thereof, wherein each ecTRX
amino acid position is numbered as in SEQ ID NO: 151. In certain embodiments, the polypeptide comprises a mutant of ecTRX that does not comprise a lysine.
In embodiments, the polypeptide further comprises a hexahistidine tag. In some embodiments the polypeptide comprises amino acids in the sequence of any one of SEQ ID
NOS:24-41 or 151.
In various embodiments, the biosensor is a pH biosensor and the ligand comprises a hydrogen ion. In embodiments, the directly responsive fluorophore is pH-sensitive. For example, the fully excited emission intensity of the directly responsive fluorophore is different at a pH less than about 7.0 compared to a pH of 7.5. In certain embodiments, the directly responsive fluorophore transitions from a monoanion to a dianion at a pH that is less than 7.0 in an aqueous solution. In some embodiments, the directly responsive fluorophore comprises a pH-sensitive fluorophore comprising fluorescein or a derivative thereof.
Exemplary Methods of Using Biosensors Provided Herein Aspects of the present subject matter provide a method of assaying for a ligand in a sample. The method may include contacting the sample with a biosensor disclosed herein under conditions such that the ligand-binding protein of the biosensor binds to the ligand if ligand is present in the sample. The method also comprises detecting (i) whether a signal is produced by a reporter group of the biosensor; and/or (ii) the a signal produced by a reporter group of the biosensor. In a non-limiting example, a reporter group of the biosensor is fluorescent, and the method further comprises contacting the reporter group with electromagnetic radiation having a wavelength that comprises a wavelength within the band of excitation wavelengths of the reporter group.
In various embodiments, the method further comprises (i) comparing a signal produced by a reporter group of the biosensor when the biosensor is contacted with the sample with a signal produced by a control sample containing a known quantity of ligand;

and (ii) detecting the presence or absence of ligand in the sample based on this comparison.
Alternatively or in addition, the method further comprises (i) comparing a signal produced by a reporter group of the biosensor when the biosensor is contacted with the sample with signals produced by a series of control samples containing known quantities of ligand; and (ii) determining the quantity of ligand in the sample based on this comparison. In some embodiments, the series of control samples comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 control samples, and wherein each control sample comprises a different quantity of ligand.
Alternatively or in addition, the method further comprises determining the concentration of a ligand in a sample, wherein determining the concentration of the ligand in the sample comprises comparing the signal to a standard hyperbolic ligand binding curve to determine the concentration of the ligand in the test sample, wherein the standard hyperbolic ligand binding curve is prepared by measuring the signal produced by the reporter group of the biosensor when the biosensor is contacted with control samples containing known concentrations of ligand. In various embodiments, the method comprises (i) measuring a ratiometric change (AR) and/or an intensity change (AI) of a signal produced by the reporter group. In some embodiments, the method includes quantitating the level of ligand present in the sample.
Aspects of the present subject matter also provide a method of assaying for multiple ligands in a sample, wherein the multiple ligands comprise a first ligand and a second ligand.
Such a method may include contacting the sample with (i) a first biosensor a first ligand provided herein and (ii) a second biosensor for the second ligand, under conditions such that the ligand-binding protein of the first biosensor binds to the first ligand, if the first ligand is present in the sample, and detecting (i) a signal produced by a reporter group of the first biosensor, or (ii) whether a signal is produced by a reporter group of the first biosensor. In some embodiments, the second biosensor is also a biosensor provided herein, and the second biosensor is contacted with the second ligand under conditions such that the ligand-binding protein of the second biosensor binds to the second ligand it is present in the sample. The method may further comprise detecting (i) a signal produced by a reporter group of the second biosensor, or (ii) whether a signal is produced by a reporter group of the second biosensor.
In some embodiments, the signal produced by the reporter group of the first biosensor is different than the signal produced by the reporter group of the second biosensor. In a non-limiting example, the reporter group of the first biosensor and the reporter group of the second biosensor are each fluorescent, and the peak emission wavelength of the reporter group of the first biosensor is at least about 10, 25, 50, 75, or 100 nm greater or lower than the peak emission wavelength of the reporter group of the second biosensor.
Non-limiting examples of biosensors include biosensors with ligand-binding proteins comprising a GGBP (e.g., an E. coli GGBP) or a derivative or mutant thereof;
(ii) an E. coli arabinose binding protein (e.g., an E. coli arabinose binding protein) or a derivative or mutant thereof; (iii) a dipeptide binding protein (e.g., an E. coli dipeptide binding protein) or a derivative or mutant thereof; (iv) a histidine binding protein (e.g., an E.
coli, histidine binding protein) or a derivative or mutant thereof; (v) a ribose binding protein (e.g., an E. coli ribose binding protein) or a derivative or mutant thereof; (vi) a sulfate binding protein (e.g., an E.
coli sulfate binding protein) or a derivative or mutant thereof; (vii) a maltose binding protein (e.g., an E. coli maltose binding protein) or a derivative or mutant thereof;
(viii) a glutamine binding protein (e.g., an E. coli glutamine binding protein) or a derivative or mutant thereof;
(ix) a glutamate/aspartate binding protein (e.g., an E. coli glutamate/aspartate binding protein) or a derivative or mutant thereof; (x) a phosphate binding protein (e.g., an E. coli phosphate binding protein) or a derivative or mutant thereof; or (xi) an iron binding protein [e.g., a Haemophilus influenza (H. influenzae) iron binding protein] or a derivative or mutant thereof. For example, the second biosensor comprises an E. coli GGBP having a YlOA, YlOC, D14C, D14A, D14Q, D14N, D14S, D14T, D14E, D14H, D14L, D14Y, D14F, N15C, Fl6L, Fl6A, Fl6C, F16Y, N91C, N91A, K92A, K92C, E93C, S112A, S115A, E149C, E149K, E149Q, E1495, H152C, H152A, H152F, H152Q, H152N, D154C, D154A, D154N, A155C, A155S, A155H, A155L, A155F, A155Y, A155N, A155K, A155M, A155W, A155Q, R158C, R158A, R158K, M182C, M182W, W183C, W183A, N211C, N211F, N211W, N211K, N211Q, N211S, N211H, N211M, N211C, D212C, L238C, D236C, D236A, D236N, L255C, N256A, N256D, D257C, P294C, and V293C mutation (e.g., comprising 1, 2, 3, 4, 5 or more of these mutations), wherein each amino acid position is numbered as in (SEQ ID
NO: 153); (ii) an E. coli arabinose binding protein having a D257C, F23C, K301C, L253C, or L298C mutation (e.g., comprising 1, 2, 3, 4, or 5 of these mutations) (see, e.g., U.S. Patent Application Publication No. 2004/0118681, the entire contents of which are incorporated herein by reference) (see, e.g., U.S. Patent Application Publication No.
2004/0118681, the entire contents of which are incorporated herein by reference); (iii) an E.
coli dipeptide binding protein having a D450C, K394C, R141C, S111C, T44C, or W315C mutation (e.g., comprising 1, 2, 3, 4, 5 or 6 of these mutations) (see, e.g., U.S. Patent Application Publication No. 2004/0118681, the entire contents of which are incorporated herein by reference); (iv) an E. coli, histidine binding protein having a E167C, K229C, V163C, Y230C, F231C, mutation (e.g., comprising 1, 2, 3, 4, 5 or 6 of these mutations) (see, e.g., U.S. Patent Application Publication No. 2004/0118681, the entire contents of which are incorporated herein by reference); (v) an E. coli ribose binding protein having a T135C, D165C, E192C, A234C, L236C, or L265C mutation (e.g., comprising 1, 2, 3, 4, 5 or 6 of these mutations) (see, e.g., U.S. Patent Application Publication No. 2004/0118681, the entire contents of which are incorporated herein by reference); (vi) an E. coli sulfate binding protein having a L65C, N70C, Q294C, R134C, W290C, or Y67C mutation (e.g., comprising 1, 2, 3, 4, 5 or 6 of these mutations) (see, e.g., U.S. Patent Application Publication No.
2004/0118681 the entire content of which is incorporated herein by reference); (vii) an E. coli maltose binding protein having a D95C, F92C, E163C, G174C, 1329C, or S233C mutation (e.g., comprising 1, 2, 3, 4, 5 or 6 of these mutations) (see, e.g., U.S. Patent Application Publication No.
2004/0118681 the entire content of which is incorporated herein by reference);
(viii) an E.
coli glutamine binding protein having a N160C, F221C, K219C, L162C, W220C, Y163C, or Y86C mutation (e.g., comprising 1, 2, 3, 4, 5 or more of these mutations) (see, e.g., U.S.
Patent Application Publication No. 2004/0118681 the entire content of which is incorporated herein by reference); (ix) an E. coli glutamate/aspartate binding protein having a A207C, A210C, El 19C, F126C, F131C, F270C, G211C, K268C, Q123C, or T129C mutation (e.g., comprising 1, 2, 3, 4, 5 or more of these mutations) (see, e.g., U.S. Patent Application Publication No. 2004/0118681 the entire content of which is incorporated herein by reference); (x) an E. coli phosphate binding protein having a A225C, N223C, N226C, 5164C, or 539C mutation (e.g., comprising 1, 2, 3, 4, or 5 of these mutations) (see, e.g., U.S.
Patent Application Publication No. 2004/0118681 the entire content of which is incorporated herein by reference); or (xi) a Haemophilus influenza (H. influenzae) iron binding protein having a E203C, K202C, K85C, or V287C mutation (e.g., comprising 1, 2, 3, or 4 of these mutations) (see, e.g., U.S. Patent Application Publication No. 2004/0118681 the entire content of which is incorporated herein by reference). In various embodiments, the sample is suspected of comprising a ligand, such as a ligand disclosed, described, or otherwise mentioned herein.

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Yao et al., Biochemistry 33: 4769-4779 (1994) Various types of samples may be used in methods provided herein. In non-limiting examples, a sample may comprise a reaction product, a buffer, and/or a solvent. In some embodiments, the solvent is an aqueous solvent. In some embodiments, the solvent comprises a non-polar solvent, a polar aprotic solvent, and/or a polar protic solvent. For example, a sample may comprise water, liquid ammonia, liquid sulfur dioxide, sulfuryl chloride, sulfuryl chloride fluoride, phosphoryl chloride, dinitrogen tetroxide, antimony trichloride, bromine pentafluoride, hydrogen fluoride, dimethyl sulfoxide, hexane, benzene, toluene, 1,4-dioxane, chlorogorm, diethyl ether, dichloromethane, N-methylpynolidone, tetrahydrofuran, ethyl acetate, acetone, dimethylformamide, acetonitrile, tormic acid, n-butanol, isopropanol, nitromethane, ethanol, methanol, and/or acetic acid.
In embodiments, a sample comprises a Newtonian liquid, a shear thickening liquid, a shear thinning liquid, a thixotropic liquid, a rheopectic liquid, or a Bingham plastic. In some implementations, a sample has a dynamic viscosity of at least about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, or 2 pascal-seconds (Pa.$) or less than about 2, 1.5, 1.4, 1.3, 1.2, 1.1, 1, 0.9, 0.8, 0.7, 0.6, 0.5 Pa=s; and/or a kinematic viscosity of at least about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, or 2 centistokes (cSt) or less than about 2, 1.5, 1.4, 1.3, 1.2, 1.1, 1, 0.9, 0.8, 0.7, 0.6, 0.5 cSt.
In various embodiments, the sample comprises a biological sample. The sample may comprise, e.g., a clinical sample (i.e., a sample collected in a clinical or veterinary setting, e.g., by or at the request or supervision or direction of a doctor, nurse, aid worker, or medic) and/or a physiological sample (a sample collected from an organism, e.g., a mammal such as a human). In certain embodiments, the biological sample comprises or has been provided or obtained from a skin surface or a mucosal surface. In some embodiments, the biological sample comprises a biological fluid. Non-limiting examples of biological fluids include sweat, tear fluid, blood, serum, plasma, interstitial fluid, amniotic fluid, sputum, gastric lavage, skin oil, milk, fecal matter, emesis, bile, saliva, urine, mucous, semen, lymph, spinal fluid, synovial fluid, a cell lysate, venom, hemolymph, and fluid obtained from plants such as the fluid transported in xylem cells or phloem sieve tube elements of a plant (e.g. sap).
The present subject matter also provides biosensors, methods, compositions, and devices useful for measuring the level of a ligand within a liquid solution or suspension or composition comprising cultured cells or tissue or a supernatant of such a solution or suspension, e.g., a sample of conditioned media or a sample of growth media in which a population of cells was cultured. In some embodiments, the sample is within a culture (e.g., inserted into a bioreactor) or provided from a media, culture, or reaction, e.g., in a bioreactor.
For example, the sample may be within or provided from a fermenter such as a culture or culture supernatant from a fermentation reaction (e.g., an ongoing fermentation). Thus, the level of a ligand can be assayed at a timepoint of interest or at a series of timepoints over the duration of cell culture, e.g. continuously, in or from a reaction or culture.
Bioreactors include devices or systems that support a biologically active environment. For example, a bioreactor may comprise a vessel in which a chemical process is carried out which involves organisms or biochemically active substances derived from such organisms. Such a process can either be aerobic or anaerobic. Organisms growing in bioreactors may be, e.g., submerged or suspended in liquid medium or may be attached to the surface of a solid medium. Submerged cultures may be suspended or immobilized. Suspension bioreactors can use a wider variety of organisms, since special attachment surfaces are not needed, and can operate at much larger scale than immobilized cultures. However, in a continuously operated process the organisms will be removed from the reactor with the effluent.
Immobilization is a general term describing a wide variety of cell or particle attachment or entrapment. It can be applied to basically all types of biocatalysis including enzymes, cellular organelles, and cells (e.g., animal cells, plant cells, fungal cells, and bacterial cells).
Immobilization is useful for continuously operated processes, since the organisms will not be removed with the reactor effluent, but is limited in scale because the cells are only present on the surfaces of the vessel.
A bioreactor may also refer to a device or system meant to grow cells or tissues in the context of cell culture. The interrogation and/or monitoring of ligand levels in such samples permits the evaluation of the status of growth of the cells or production of secreted products by the cells to inform harvest or feeding or other modification of the culture.
Aspects of the present subject matter relate to the use of methods and biosensors provided herein to detect contamination.
In some embodiments, the sample comprises an environmental sample. Depending on context, there are instances in which a biological sample may also be, or may be within, an environmental sample. In certain embodiments, an environmental sample comprises a solute obtained from a biological composition, such as bone, nail, hair, shell, or cartilage. In various embodiments, an environmental sample comprises a solute obtained from an environmental substance and/or an environmental surface. For example, the solute may be dissolved/obtained from the environmental substance and/or an environmental surface using an aqueous or nonaqueous solution. In some embodiments, an aqueous may optionally comprise a nonaqueous solvent (e.g., mixed with an aqueous solvent). Non-limiting examples of environmental substances include rock, soil, clay, sand, meteorites, asteroids, dust, plastic, metal, mineral, fossils, sediment, and wood. Non-limiting examples of environmental surfaces include the surface of a vehicle such as a civilian vehicle (e.g., a satellite, a bike, a rocket, an automobile, a truck, a motorcycle, a yacht, a bus, or a plane) or a military vehicle (e.g., a tank, an armored personnel carrier, a transport truck, a jeep, a mobile artillery unit, a mobile antiaircraft unit, a minesweeper, a Mine-Resistant Ambush Protected (MRAP) vehicle, a lightweight tactical all-terrain vehicle, a high mobility multipurpose wheeled vehicle, a mobile multiple rocket launch system, an amphibious landing vehicle, a ship, a hovercraft, a submarine, a transport plane, a fighter jet, a helicopter, a rocket, or an Unmanned Arial Vehicle), a drone, a robot, a building, furniture, or an organism other than a human. In some embodiments, the sample comprises an environmental fluid. Non-limiting examples of environmental fluids include marine water, well water, drinking well water, water at the bottom of well dug for petroleum extraction or exploration, melted ice water, pond water, aquarium water, pool water, lake water, mud, stream water, river water, brook water, waste water, treated waste water, reservoir water, rain water, and ground water. In some embodiments, waste water comprises sewage water, septic tank water, agricultural runoff, water from an area in which chemical or oil spill has or is suspected of having occurred (e.g., an oil spill into a marine environment), water from an area where a radiation leak has or is suspected of having occurred (e.g., coolant from a nuclear reactor), water within the plumbing of a building, water within or exiting a research facility, and/or water within or exiting a manufacturing facility such as a factory.
As used herein, "suspected" with respect to an event means that there has been at least one test (e.g., a test other than a method or assay provided herein), occurrence (e.g., that is likely to or that may cause the event such as an emergency, leak, accident, flood, earthquake, storm, fire, malfunction, sunk vessel, or crash), or report (e.g., by a witness, informant, or observer) that is consistent with the event having occurred.
In certain embodiments, the sample comprises a food or beverage additive and/or a food or beverage composition. In some embodiments, the food or beverage composition comprises a fermented composition. In various embodiments, the sample comprises a fluid obtained from a food composition. Alternatively or in addition, the sample may comprise a solute dissolved from a food composition. In some examples, a solute is or has been dissolved from a food composition with an aqueous or nonaqueous solution. In various implementations, an aqueous solution may optionally comprise a nonaqueous solvent. In certain embodiments, a sample comprises a food composition in semisolid or liquid form.
Non-limiting examples of such compositions include yogurt, soup, ice cream, a broth, a puree, a shake, a smoothie, a batter, a condiment, a sauce, and any combination thereof. In some implementations, a sample is a food engineering process (e.g., obtained from a food design, storage, transport, or production process or from equipment intended to process, transport, or store food). A food composition may comprise, e.g., a plant or a composition isolated from a plant, and/or an animal or a composition isolated from an animal. In various embodiments, a sample comprises a beverage composition. Non-limiting examples of beverage compositions include soft drinks, fountain beverages, water, coffee, tea, milk, dairy-based beverages, soy-based beverages (e.g., soy milk), almond-based beverages (e.g., almond milk), vegetable juice, fruit juice, fruit juice-flavored drinks, energy drinks, sports and fitness drinks, alcoholic products, and beverages comprising any combination thereof.
Non-limiting examples of beverage compositions comprising water include purified water (e.g., filtered water, distilled water, or water purified by reverse osmosis), flavored water, mineral water, spring water, sparkling water, tonic water, and any combination thereof. In various embodiments, the sample comprises alcohol. Non-limiting examples of such samples include samples comprising or obtained/provided from beer, malt beverages, liqueur, wine, spirits, and any combination thereof.
In some embodiments, a sample comprises a nutritional or supplement composition.
In certain implementations, the nutritional or supplement composition comprises an omega-3 fatty acid, a vitamin, a mineral, a protein powder, or a meal supplement.
In certain embodiments, a biosensor is implanted in a subject's body. For example, a biosensor may be implanted in a subject's blood vessel, vein, eye, natural or artificial pancreas, alimentary canal, stomach, intestine, esophagus, or skin (e.g., within the skin or under the skin). In various embodiments, the biosensor is configured within or on the surface of a contact lens. In some embodiments, the biosensor is configured to be implanted in or under the skin. In non-limiting examples, the biosensor is implanted in a subject with an optode and/or a microbead. In certain embodiments, the biosensor generates a signal transdermally.
Aspects of the present subject matter provide a method for assaying the level of ligand in a subject. The method may comprise contacting a biological sample from the subject with a biosensor for ligand under conditions such that the biosensor binds to ligand present in the biological sample. The biosensor comprises reporter group that is attached to a ligand binding protein, and binding of ligand to a ligand-binding domain of the ligand binding protein causes a change in signaling by the reporter group.
In various embodiments, the subject has or is suspected of having a disease or disorder, such as abnormal kidney function, abnormal adrenal gland function, diabetes, hypochloremia, bromism, hypothyroidism, hyperthyroidism, cretinism, depression, fatigue, obesity, a low basal body temperature, a goiter, a fibrocystic breast change, lactic acidosis, septic shock, carbon monoxide poisoning, asthma, a lung disease, respiratory insufficiency, Chronic Obstructive Pulmonary Disease (COPD), regional hypoperfusion, ischemia, severe anemia, cardiac arrest, heart failure, a tissue injury, thrombosis, or a metabolic disorder, diarrhea, shock, ethylene glycol poisoning, methanol poisoning, diabetic ketoacidosis, hypertension, Cushing syndrome, liver failure, cancer, or an infection.
As used herein, "suspected" with respect to a subject's condition (e.g., disease or injury) means that the subject has at least one symptom or test (e.g., a test other than an assay or method provided herein) that is consistent with the condition.
In some embodiments, the biological sample comprises blood, plasma, serum, sweat, tear fluid, or urine. In certain embodiments, the biological sample is present in or on the surface of the subject. In various implementations, the biosensor is applied onto or inserted into the subject. For example, the biosensor may be tattooed into the subject or is in or on a device that is implanted into the subject. In some embodiments, the biosensor may be present in or on a contact lens that is worn by the subject. Methods for determining the level of ligand in a subject who has or is suspected of having a disease that results in or from (or otherwise involves) an altered level of a ligand, may be performed without other testing related to the disease performed as part of a battery of clinical testing.
The present subject matter includes a method for monitoring the level of a ligand, comprising periodically or continuously detecting the level of the ligand, wherein detecting the level of the ligand comprises (a) providing or obtaining a sample; (b) contacting the sample with a biosensor for the ligand under conditions such that the ligand-binding protein of the biosensor binds to the ligand, and (c) detecting a signal produced by the biosensor.
Aspects of the present subject matter also provide a method for monitoring the level of a ligand in a subject, comprising periodically detecting the level of the ligand in the subject. Detecting the level of the ligand in the subject may comprise (a) providing or obtaining a biological sample from the subject; (b) contacting the biological sample with a biosensor for the ligand provided herein under conditions such that the ligand-binding protein of the biosensor binds to the ligand, if the ligand is present in the biological sample, and (c) detecting (i) a signal produced by a reporter group of the biosensor, or (ii) whether a signal is produced by a reporter group of the biosensor. The level of the ligand may be detected, e.g., at least once every 1, 2, 3, 6, or 12 hours, at least once every 1, 2, 3, or 4 days, at least once every 1, 2, or three weeks, or at least once every 1, 2, 3, 4, 6, or 12 months.
The present subject matter also provides a method for monitoring the level of a ligand in a subject. The method comprises (a) administering a biosensor provided herein or a device comprising a biosensor provided herein to the subject, wherein after administration the biosensor is in contact with a bodily fluid or surface that typically comprises the ligand, and (b) detecting (i) a signal produced by a reporter group of the biosensor continuously or repeatedly at intervals less than about 30 minutes (m), 15m, 10m, 5m, lm, 30 seconds (s), 15s, 10s, 5s, ls, 0.1s, 0.001s, 0.0001s, or 0.00001 apart, and/or (ii) whether a signal is produced by a reporter group of the biosensor continuously or repeatedly at intervals less than about 30m, 15m, 10m, 5m, lm, 30s, 15s, 10s, 5s, ls, 0.1s, 0.001s, 0.0001s, or 0.00001apart.
Non-limiting aspects of continuously monitoring ligand levels are described in Weidemaier et al. (2011) Biosensors and Bioelectronics 26, 4117-4123 and Judge et al.
(2011) Diabetes Technology & Therapeutics, 13(3):309-317, the entire contents of each of which are hereby incorporated herein by reference.
Also within the invention is a composition comprising a purified thermostable, ligand-binding fluorescently-responsive sensor protein and a solid substrate, e.g., a particle, a bead such as a magnetic bead, or a planar surface such as a chip or slide, wherein the sensor protein is immobilized onto the solid substrate. An exemplary solid substrate solid substrate comprises a cyclic olefin copolymer.
A thermostable ligand sensor protein is one in which the activity (ligand binding) is retained after exposure to relatively high temperatures. For example, the ligand sensor protein comprises a mid-point thermal melt transition greater than 50 C, greater than 60 C, greater than 70 C, greater than 80 C, greater than 90 C, or greater than 100 C. In some embodiments, the sensor protein contains a single cysteine residue. In some embodiments, the single cysteine residue is located in a site of the ligand-binding protein, where it responds to ligand binding. In some examples, the protein comprises the amino acid sequence of SEQ
ID NO: 16 (ttGGBP.17C.O.bZif) and 19 (ttGGBP.182C.O.bZif), and in some examples, the single cysteine is conjugated to Badan, Acrylodan, or a derivative thereof.
For example, the derivative comprises a replacement of the two-ring naphthalene of Acrylodan or Badan with a three-ring anthracene, a fluorene, or a styrene. A reporter group is covalently bound to the single cysteine. In some situations, the solid substrate comprises a plurality of sensor proteins, each of which comprises a different dissociation constant (K ) for ligand, e.g., for detecting and quantifying ligand levels across many ranges of concentrations.
The present subject matter also includes a composition comprising purified ligand sensor protein with less than 65% identity and greater than 27% identity (e.g., 44-48%
sequence identity) to any ligand-binding protein disclosed herein, wherein the sensor protein comprises a single cysteine residue, such that the sensor protein is immobilized onto the solid substrate. As described above, a reporter group is covalently bound to the single cysteine. In some example, the solid substrate comprises a plurality of sensor proteins, each of which comprises a different dissociation constant (Kd) for ligand for sensing over a wide range or ranges of ligand concentrations.
In some embodiments, a method of detecting the presence of or the quantity of ligand in a test sample is carried out using the following steps: contacting the test sample with the biosensor or sensor protein/solid support construct to yield a complex of ligand and the ligand-binding protein or biosensor protein; contacting the complex with an excitation light;
measuring an emission intensity of the reporter group from at least two wavelengths;
computing a ratiometric signal from the two (or more) wavelengths; and comparing the signal to a known ligand binding curve of signals to identify the presence of or calculate the quantity of ligand in the test sample. The test sample may be obtained from a variety of sources. For example, the test sample may be selected from a bodily fluid, a food, a beverage, or a bioreactor culture broth. The testing method may be carried out in vivo, e.g., using an implantable device or dermal patch, or ex vivo.
In various embodiments, the subject to be tested is a mammal, e.g., a primate (such as a human, a monkey, a chimpanzee, or a gorilla), a fish, a bird, a reptile, an amphibian, or an arthropod. In some embodiments, the subject is a fish, a cow, a pig, a camel, a llama, a horse, a race horse, a work horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a wolf, a dog (e.g., a pet dog, a work dog, a police dog, or a military dog), a rat, a mouse, a seal, a whale, a manatee, a lizard, a snake, a chicken, a goose, a swan, a duck, or a penguin.
In some embodiments, the ligand comprises a halide anion and the ligand-binding protein comprises a fluorescent protein. Aspects of the present subject matter provide a method for detecting the level of a halide anion in a sample, comprising contacting the sample with a biosensor for a halide anion under conditions such that the biosensor binds to a halide anion present in the sample. In various embodiments the biosensor comprises a halide anion-binding fluorescent protein, and binding of the halide anion to a halide anion-binding domain of the fluorescent protein causes a change in signaling by the fluorescent protein. In some embodiments, the sample is an environmental sample. In a non-limiting example, the sample comprises treated wastewater or drinking water.
Aspects of the present subject matter further provides a method for assaying the level of chloride in a subject, comprising contacting a biological sample from the subject with a biosensor for chloride under conditions such that the biosensor binds to chloride present in the biological sample. The biosensor may comprise, e.g., a chloride-binding fluorescent protein, and binding of chloride to a chloride-binding domain of the fluorescent protein causes a change in signaling by the fluorescent protein. In some embodiments, the subject has or is suspected of having hypochloremia. Alternatively or in addition, the subject has or is suspected of having abnormal kidney or adrenal gland function. In certain embodiments, the biological sample comprises blood, plasma, serum, sweat, tear fluid, or urine. In some embodiments, the method is performed as part of a battery of clinical testing.
Also provided is a method for assaying the level of iodide in a subject, comprising contacting a biological sample from the subject with a biosensor for iodide under conditions such that the biosensor binds to iodide present in the biological sample, wherein the biosensor comprises an iodide-binding fluorescent protein. Binding of iodide to an iodide-binding domain of the fluorescent protein causes a change in signaling by the fluorescent protein. In some embodiments, the subject has or is suspected of having hypothyroidism, hyperthyroidism, cretinism, depression, fatigue, obesity, a low basal body temperature, a goiter, or a flbrocystic breast change. In some embodiments, the biological sample comprises blood, plasma, serum, sweat, tear fluid, or urine. In some embodiments, the method is performed as part of a battery of clinical testing.
The present subject matter further includes a method for assaying the level of bromide in a subject, comprising contacting a biological sample from the subject with a biosensor for bromide under conditions such that the biosensor binds to bromide present in the biological sample, wherein the biosensor comprises a bromide-binding fluorescent protein.
Upon binding of bromide to a bromide-binding domain of the fluorescent protein, the signal of the fluorescent protein changes. In some embodiments, the subject has or is suspected of having bromism. In certain embodiments, the biological sample comprises blood, plasma, serum, sweat, tear fluid, or urine. In some embodiments, the method is performed as part of a battery of clinical testing.
Exemplary Devices and Compositions Comprising Biosensors Aspects of the present subject matter provide a device comprising one or more biosensors provided herein. Such devices may be, e.g., wearable, implantable, portable, or fixed.
In some embodiments, the device is a nanoparticle or a microparticle comprising the biosensor. Non-limiting examples of devices include devices comprising a test strip, patch, plate, bead, or chip comprising a biosensor provided herein. In certain embodiments, a device may comprise a desiccated biosensor.
The present subject matter also provides a contact lens or a skin patch comprising a biosensor provided herein. In some embodiments, the biosensor is throughout the contact lens or skin patch or within a particular region or zone of a contact lens or skin patch (e.g., in one or more shapes (e.g., a square, circle, or star), dots, lines, or zones, located at the periphery or a portion of the periphery of a contact lens or patch). In some embodiments, the skin patch comprises an adhesive that facilitates attachment of the patch to the surface of skin.
Devices provided herein may include a variety of structural compositions. For example, many polymers (including copolymers), and plastics may be used. Non-limiting examples of compositions useful in certain devices include glass, polystyrene, polypropylene, cyclic olefin copolymers, ethylene-norbomene copolymers, polyethylene, dextran, nylon, amylase, paper, a natural cellulose, a modified cellulose, a polyacrylamide, gabbros, gold, and magnetite (as well as combinations thereof). In some embodiments, the device comprises a hydrogel, a cryogel, or a soluble gel. For example, the biosensor may be incorporated into or onto the hydrogel, cryogel, or soluble gel. In various embodiments, the device comprises a matrix comprising nanopores, micropores, and/or macropores.
In certain embodiments, the surface of a device comprises a polymer. In an embodiment, the surface comprises the surface of a particle or a bead having a diameter of about 0.001-1, 0.001-0.1, 0.01-0.1, 0.001-0.01, 0.1-1, 0.1-0.5, or 0.01-0.5 centimeters (cm). For example, the particle comprises a nanoparticle or a microparticle.

Non-limiting examples of polymers include cyclic olefin copolymers, ethylene-norbomene copolymers, polylactic acid, polyglycolic acid, agarose, alginate, poly(lactide-co-glycolide), gelatin, collagen, agarose, natural and synthetic polysaccharides, polyamino acids, poly(lysine), polyesters, polyhydroxybutyrates, polyanhydrides, polyphosphazines, polyvinyl alcohol, polyalkylene oxide, polyethylene oxide, polyallylamines, polyacrylates, modified styrene polymers, poly(4-aminomethylstyrene), pluronic polyols, polyoxamers, polyuronic acid, polyvinylpynolidone, hydroxyethyl (meth)acrylate, polyolefins, polyurethane, polystyrene, ethylene/methacrylic acid copolymers, ethylene/methyl methacrylate copolymers, polyester, and polyurethane. In some embodiments, the patch comprises a woven fabric, a knitted fabric, or a nonwoven fabric of a synthetic fiber and/or natural fiber.
Non-limiting examples of temporary tattoo compositions for application to a subject's skin are discussed in U.S. Patent Application Publication No. 20090325221, published December 31, 2009, and U.S. Patent No. 6,428,797, the entire contents of each of which are incorporated herein by reference. Biosensor disclosed herein may be incorporated into any temporary tattoo or other composition for application to the skin. For example, a temporary tattoo decal for application to a subject's skin and configured to detect the presence of a ligand may comprise, e.g., a base paper or plastic; a water-soluble slip layer applied to the base paper or plastic; a temporary tattoo applied to the water-soluble release layer on the base paper, wherein the temporary tattoo comprises a biosensor disclosed herein; an adhesive layer overlying the temporary tattoo; and a protective sheet overlying the adhesive layer.
In some embodiments, the device comprises a plastic polymer comprising cyclic olefin copolymer (COC), such as e.g. TOPAS COC. Several types of cyclic olefin copolymers are available based on different types of cyclic monomers and polymerization methods. Cyclic olefin copolymers are produced by chain copolymerization of cyclic monomers such as 8,9,10-trinorbom-2-ene (norbomene) or 1,2,3,4,4a,5,8,8a-octahydro-1,4:5,8-dimethanonaphthalene (tetracyclododecene) with ethene (such as TOPAS
Advanced Polymer's TOPAS, Mitsui Chemical's APEL), or by ring-opening metathesis polymerization of various cyclic monomers followed by hydrogenation (Japan Synthetic Rubber's ARTON, Zeon Chemical's Zeonex and Zeonor). See, e.g., International Union of Pure and Applied Chemistry (2005) Purr. AppL Chem. 77(5):801-814. These later materials using a single type of monomer may be referred to as cyclic olefin polymers (COPs). A CAS Registry number for COC is 26007-43-2.

In certain embodiments, the device is attached to a surface of a device or is not attached to a surface of the device (e.g., the biosensor is present loosely within the device as a component of a solution or powder).
A biosensor may be attached to a device via a variety or means, e.g., via attachment motif. In some embodiments, the attachment motif is attached to the N-terminus or the C-terminus of the biosensor. In certain embodiments, the biosensor is linked to an attachment motif via a covalent bond. In various embodiments, the biosensor is linked to the attachment motif via a linker. A non-limiting example of a linker is a polyglycine comprising 2, 3, 4, 5, or more glycines and optionally further comprising a serine. In some embodiments, the attachment motif comprises a polypeptide. Non-limiting examples of polypeptides useful in attachment moieties include hexahistidine peptides, hexalysine peptides, zinc-finger domains (ZF-QNKs), and disulfide-containing truncated zinc fingers (f3Zifs). An example of a hexalysine peptide comprises amino acids in the sequence of SEQ ID NO: 45, an example of a ZF-QNK comprises amino acids in the sequence of SEQ ID NO: 43, and an example of a PZif comprises amino acids in the sequence of SEQ ID NO: 42. In some embodiments, the attachment motif comprises a polypeptide that binds to plastic or cellulose.
The hexahistidine, hexalysine, PZif and QNK-ZF fusions enable FRSs to be immobilized onto chemically functionalized surfaces. Non-limiting aspects of chemically functionalized surfaces are discussed in Biju, V. (2014) Chem Soc Rev, 43, 744-64 and McDonagh (2008) Chem Rev, 108, 400-422, the entire contents of which are incorporated herein by reference. Directed evolution methods have been used to develop peptides that bind directly to non-functionalized surfaces (Care, Bergquist and Sunna 2015 Trends Biotechnol, 33, 259-68; Baneyx 2007 Curr. Opin. Biotechnol., 18, 312-317;
Gunay and Klok 2015 Bioconjug Chem, 26, 2002-15), including various plastics (Adey et al.
1995 Gene, 156, 27-31; Serizawa et al. 2005 J Am Chem Soc, 127, 13780-1; Serizawa, Sawada and Kitayama 2007a Angew Chem Int Ed Engl, 46, 723-6; Serizawa, Sawada and Matsuno 2007b Langmuir, 23, 11127-33; Serizawa, Techawanitchai and Matsuno 2007c Chembiochem, 8, 989-93; Matsuno et al. 2008 Langmuir, 24, 6399-403; Chen, Serizawa and Komiyama 2011 J
Pept Sci, 17, 163-8; Kumada 2010 J. Biosci. and BioEng., 109, 583-587; Date et al. 2011 ACS Appl Mater Interfaces, 3, 351-9; Kumada 2012, Vodnik, Strukelj and Lunder 2012 J.
Biotech., 160, 222-228; Kumada 2014 Biochem. et Biophys. Acta, 1844, 1960-1969; Ejima, Matsuno and Serizawa 2010 Langmuir, 26, 17278-85), inorganic materials(Hnilova 2012 Soft Matter, 8, 4327-4334; Care et al. 2015 Trends Biotechnol, 33, 259-68), nanoparticles (Avvakumova et al. 2014 Trends Biotechnol, 32, 11-20), and cellulosic paper (Guo et al.
2013 Biomacromolecules, 14, 1795-805). Such peptides, or natural material-binding domains (Oliveira et al. 2015 Biotechnol Adv, 33, 358-69), also can be fused to FRSs to direct site-specific, oriented immobilization on their target materials while preserving FRS function.
For instance, plastic-binding peptides have been developed that direct immobilization on polystyrene (Adey et al. 1995 Gene, 156, 27-31; Serizawa et al. 2007c Chembiochem, 8, 989-93; Kumada 2010 Biochem. et Biophys. Acta, 1844, 1960-1969; Vodnik et al. 2012 Anal Biochem, 424, 83-6), polymethyl acrylate (Serizawa et al. 2005 J Am Chem Soc, 127, 13780-1; Serizawa et al. 2007a Angew Chem Int Ed Engl, 46, 723-6; Serizawa et al.
2007b Langmuir, 23, 11127-33; Kumada 2014 Biochem. et Biophys. Acta, 1844, 1960-1969), polycarbonate (Kumada 2012 J. Biotech., 160, 222-228), polylactide (Matsuno et al. 2008 Langmuir, 24, 6399-403), and polyphenylene vinylene (Ejima et al. 2010 Langmuir, 26, 17278-85). Cellulose-binding peptides (Guo et al. 2013 Biomacromolecules, 14, 1795-805) and natural domains (Oliveira et al. 2015 Biotechnol Adv, 33, 358-69;
Shoseyov, Shani and Levy 2006 Microbiol Mol Biol Rev, 70, 283-95) can be used to immobilize fusion proteins on paper. Inorganic material include noble metals (Hnilova 2012 Soft Matter, 8, 4327-4334), semi-conductors (Care et al. 2015 Trends Biotechnol, 33, 259-68), and fluorescent quantum dots(Medintz et al. 2005 Nat Mater, 4, 435-46; Lee et al. 2002 Science, 296, 892-5). The entire contents of each of the references above (and all other references herein) is incorporated herein by reference.
In some embodiments, the attachment motif is attached to a device surface and/or within a matrix of the device. In some embodiments, a biosensor is attached to an attachment motif via a covalent bond and the attachment motif is attached to a device via a covalent bond. Non-limiting examples of covalent bonds include disulfide bonds, ester bonds, thioester bonds, amide bonds, and bonds that have been formed by click reactions. Non-limiting examples of a click reaction include a reaction between an azide and an alkyne; an azide and an alkyne in the presence of Cu(I); an azide and a strained cyclooctyne; an azide and a dibenzylcyclooctyne, a difluorooctyne, or a biarylazacyclooctynone; a diaryl-strained-cyclooctyne and a 1,3-nitrone; an azide, a tetrazine, or a tetrazole and a strained alkene; an azide, a tetrazine, or a tretrazole and a oxanorbomadiene, a cyclooctene, or a trans-cycloalkene; a tetrazole and an alkene; or a tetrazole with an amino or styryl group that is activated by ultraviolet light and an alkene.

Alternatively or in addition, a surface of a device may be modified to contain a moiety (e.g. a reactive group) what facilitates the attachment of a biosensor and/or binds to the biosensor. In some embodiments, the biosensor is attached to a surface via a biotin-avidin interaction.
In various implementations, the device comprises a first region for receiving a sample and second a region that comprises the biosensor, wherein the first region is separated from the second region by a filter. In some examples, the filter is impermeable to compounds greater than about 1, 2, 3, 4, 5, 10, 50, 200, or 250 kiloDalton (kDa) in size. The sample may comprise, e.g., a tube, such as a tube that is configured for centrifugation.
When sample is placed into the first region and the device is centrifuged, then a portion of the sample comprising a ligand flows through the filter into the second region where the biosensor is contacted.
Non-limiting examples of devices provided herein include endoscopy probes and colonoscopy probes.
In some embodiments, the device comprises an optode. In non-limiting examples, the optode comprises an optical fiber and a single biosensor or composite biosensor. In certain embodiments, the single biosensor or composite biosensor is immobilized on the surface or at an end of the optical fiber. In some embodiments, the optode is configured for implantation into a subject. Alternatively or in addition, the optode is configured for insertion into a sample.
The devices provided herein may optionally comprise a biosensor panel, a composite sensor, a sensor array, and/or a composition comprising a plurality of biosensors. In various embodiments, a device comprises multiple ligand biosensors that detect a range of different ligand concentrations in a single sample and/or assay run (i.e., each biosensor has a different affinity for ligand). Devices may provide spatial localization of multiple biosensors to provide the necessary addressability of different elements in a multi-sensor array comprising sensors that differ in their engineered affinities for coverage of a wide range of ligand concentrations, or sensors that each detects distinct analytes.
Aspects of the present subject matter provide a biosensor panel comprising a plurality of biosensors, wherein the plurality of biosensors comprises at least one biosensor disclosed herein. In some embodiments, the plurality comprises at least about 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 biosensors.

The present subject matter also provides a composite sensor. The composite sensor may comprise a sensor element, wherein the sensor element comprises 2 or more biosensors, wherein at least 1 of the 2 or more biosensors is a biosensor disclosed herein. In some embodiments, the biosensors are not spatially separated in the sensor element, e.g., the biosensors are mixed within a solution or on a surface of the sensor element.
In various embodiments, the composite sensor comprises a plurality of sensor elements, wherein each sensor element of the plurality of sensor elements comprises 2 or more biosensors, wherein at least 1 of the 2 or more biosensors is a biosensor provided herein. In some embodiments, the plurality of sensor elements comprises at least about 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 sensor elements.
Also included herein is a sensor array comprising a plurality of biosensors of the present subject matter. The sensor array may include, e.g., multichannel array or a multiplexed array. In some embodiments, the biosensors of the plurality of biosensors are spatially separated from each other. In certain embodiments, the biosensors are arranged linearly or in a grid on a surface of the array.
The present subject matter provides a composition comprising a plurality of biosensors including at least one biosensor disclosed herein. Also provided is a non-human mammal comprising a biosensor or device disclosed herein.
Exemplary Polypeptides and Polynucleotides The present subject matter provides polynucleotides encoding any one of the polypeptides disclosed herein. The polypeptides are also provided. In various embodiments, the polynucleotides are codon-optimized for expression in a desired host cell, such as bacterial cells (e.g., E. coli), yeast, insect cells, plant cells, algal cells, or mammalian cells.
The polypeptides provided herein include polypeptides comprising the amino acid sequence of any one of SEQ ID NOS: 1-41, 87-151, or 153-167. The polynucleotides provided herein include polynucleotides encoding a polypeptide comprising the amino acid sequence of any one of SEQ ID NOS: 1-41, 87-151, or 153-167.
The polypeptides and biosensors provided herein may be in a variety of forms, e.g., purified in solution, dried (e.g. lyophilized) such as in the form of a powder, and in the form of a crystal (e.g., a crystal suitable for x-ray crystallography). Thus, aspects of the present subject matter provide crystal structures and crystalized forms of the ligand-binding proteins and biosensors disclosed herein. Such crystal structures and crystalized proteins are useful for designing and optimizing biosensors using principles and methods discussed herein.
Also provided are expression vectors comprising a polynucleotide of the present subject matter and/or encoding a polypeptide disclosed herein. Non-limiting examples of expression vectors include viral vectors and plasmid vectors. In some embodiments, an expression vector comprises nucleotides in the sequence set forth as any one of SEQ ID
NOS: 46-86. In various embodiments, a polynucleotide encoding a ligand-binding protein and/or biosensor is operably linked to a promoter. The promoter may be expressed, e.g., in a prokaryotic and/or a eukaryotic cell.
The subject matter further includes an isolated cell comprising an expression vector provided herein. The isolated cell may be, e.g., a bacterial cell, a yeast cell, an algal cell, a plant cell, an insect cell, or a mammalian cell. Also included is a non-human multicellular organism such as a plant or an animal (e.g., an insect, a mammal, a worm, a fish, a bird, or a reptile) comprising an expression vector disclosed herein.
Exemplary Methods for Designing Biosensors Aspects of the present subject matter provide method of identifying a candidate ligand-binding protein for use in a biosensor, comprising: (a) selecting a first protein having a known amino acid sequence (seed sequence), wherein the first protein is a ligand binding protein; (b) identifying a second protein having an amino acid sequence (hit sequence) with at least 15% sequence identity to the seed sequence; (c) aligning the seed amino acid sequence and the hit sequence, and comparing the hit sequence with the seed sequence at positions of the seed sequence that correspond to at least 5 primary complementary surface (PCS) amino acids, wherein each of the at least 5 PCS amino acids has a hydrogen bond interaction or a van der Waals interaction with ligand when ligand is bound to the first protein; and (d) identifying the second protein to be a candidate ligand-binding protein if the hit sequence comprises at least 5 amino acids that are consistent with the PCS.
The present subject matter also includes a method for constructing a candidate biosensor, comprising: (a) providing a candidate ligand-binding protein; (b) generating a structure of the second protein; (c) identifying at least one putative allosteric, endosteric, or peristeric site of the second protein based on the structure; (d) mutating the second protein to substitute an amino acid at the at least one putative allosteric, endosteric, or peristeric site of the second protein with a cysteine; and (e) conjugating a fluorescent compound to the cysteine. In some embodiments, the structure comprises a homology model of the second protein generated using a structure of the first protein. In some embodiments, the structure comprises a structure experimentally determined by nuclear magnetic resonance spectroscopy or X-ray crystallography.
Aspects of the present subject matter further provide a method for constructing a biosensor comprising a desired dissociation constant (Kd) for ligand, comprising: (a) providing an initial biosensor that does not comprise the desired Kd for ligand, wherein the initial biosensor is a biosensor provided herein; (b) mutating the initial biosensor to (i) alter a direct interaction in the PCS between the initial biosensor and bound ligand;
(ii) manipulate the equilibrium between open and closed states of the initial biosensor; (iii) alter an interaction between the ligand-binding protein and the reporter group of the initial biosensor;
or (iv) alter an indirect interaction that alters the geometry of the binding site of the biosensor, to produce a modified biosensor; and (c) selecting the modified biosensor if the modified biosensor comprises the desired Kd for ligand. In some embodiments, the reporter comprises Acrylodan, Badan, or a derivative thereof, and mutating the initial biosensor in (b) comprises altering an interaction between the ligand-binding protein and a carbonyl group of the Acrylodan, Badan, or derivative thereof. In some embodiments, the reporter group comprises Acrylodan, Badan, or a derivative thereof, and mutating the initial biosensor in (b) comprises altering an interaction between the ligand-binding protein and a naphthalene ring of the Acrylodan, Badan, or derivative thereof. In some embodiments, mutating the initial biosensor comprises introducing a substitution mutation into the initial biosensor. In some embodiments, the method further comprises immobilizing the affinity-tuned biosensor on a substrate.
In some embodiments, the second protein comprises (i) amino acids in the sequence of any one of SEQ ID NOS: 1-41, 87-151, or 153-167; (ii) a stretch of amino acids in a sequence that is least about 95, 96, 97, 98, or 99% identical to the sequence of any one of SEQ ID NOS: 1-41, 87-151, or 153-167; (iii) a stretch of at least about 50, 100, 150, 200, 250, 300, 350, or 400 amino acids in a sequence that is at least about 95, 96, 97, 98, or 99%
identical to a sequence within any one of SEQ ID NOS: 1-41, 87-151, or 153-167; or (iv) a stretch of at least about 50, 100, 150, 200, 250, 300, 350, or 400 amino acids in a sequence that is identical to a sequence within any one of SEQ ID NOS: 1-41, 87-151, or 153-167. In various embodiments, attaching the reporter group to the putative allosteric, endosteric, or peristeric site of the first protein comprises substituting a cysteine at the site with a cysteine.

For example, the reporter group is conjugated to the cysteine. Preferably, attaching a reporter group to the corresponding amino acid of the second protein produces a functional biosensor.
Aspects also provide method for constructing a biosensor, comprising (a) providing a ligand-binding protein; (b) identifying at least one putative allosteric, endosteric, or peristeric site of the ligand-binding based a structure of the ligand-binding protein;
(c) mutating the ligand-binding protein to substitute an amino acid at the at least one putative allosteric, endosteric, or peristeric site of the second protein with a cysteine; (d) conjugating a donor fluorophore or an acceptor fluorophore to the cysteine to produce single labeled biosensor;
(e) detecting whether there is a spectral shift or change in emission intensity of the single labeled biosensor upon ligand binding when the donor fluorophore or the acceptor fluorophore is fully excited; and (f) if a spectral shift or change in emission intensity is detected in (g), attaching a donor fluorophore to the second protein if an acceptor fluorophore is attached to the cysteine, and attaching an acceptor fluorophore to the second protein if an acceptor fluorophore is attached to the cysteine.
In some embodiments, the ligand-binding protein has been identified by (i) selecting a first protein having a known amino acid sequence (seed sequence), wherein the first protein is a ligand-binding protein; (ii) identifying a second protein having an amino acid sequence (hit sequence) with at least 15% sequence identity to the seed sequence; (iii) aligning the seed amino acid sequence and the hit sequence, and comparing the hit sequence with the seed sequence at positions of the seed sequence that correspond to at least 5 primary complementary surface (PCS) amino acids, wherein each of the at least 5 PCS
amino acids has a hydrogen bond interaction or a van der Waals interaction with ligand when ligand is bound to the first protein; and (iv) identifying the second protein to be a ligand-binding protein if the hit sequence comprises at least 5 amino acids that are consistent with the PCS.
The spectral shift may comprise, e.g., a monochromatic fluorescence intensity change or a dichromatic spectral shift.
Also provided is a method of converting a biosensor that shows a monochromatic response upon ligand binding into a biosensor with a dichromatic response upon ligand binding, the method comprising (a) selecting a biosensor that exhibits a monochromatic response upon ligand binding, wherein the biosensor comprises a ligand-binding protein and a first reporter group; and (b) attaching a second reporter group to the biosensor, wherein the second reporter group has (i) an excitation spectrum that overlaps with the emission spectrum of the first reporter group; or (ii) an emission spectrum that overlaps with the excitation spectrum of the first reporter group. In certain embodiments, the second reporter group is within about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 4, 6, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, or 200 angstroms (A) of the first reporter group regardless of whether ligand is bound to the biosensor. In some embodiments, when the ligand is bound to the biosensor, the average distance between the first reporter group and the second reporter group changes by less than about 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, or 0.01 angstroms (A) compared to when ligand is not bound to the ligand-binding protein.
Aspects include a method of converting a biosensor that shows a monochromatic response upon ligand binding into a biosensor with a dichromatic response upon ligand binding, the method comprising (a) selecting a biosensor that exhibits a monochromatic response upon ligand binding, wherein the biosensor comprises a ligand-binding fluorescent protein; and (b) attaching an acceptor fluorophore or a donor fluorophore to the biosensor, wherein (i) the acceptor fluorophore has an excitation spectrum that overlaps with the emission spectrum of the fluorescent protein; or (ii) the donor fluorophore has an emission spectrum that overlaps with the excitation spectrum of the fluorescent protein.
Also provided is a method of increasing a dichromatic response of a biosensor to ligand binding, the method comprising (a) selecting a biosensor that exhibits a dichromatic response upon ligand binding, wherein the biosensor comprises a ligand-binding protein and a first reporter group; and (b) attaching a second reporter group to the biosensor, wherein the second reporter group has (i) an excitation spectrum that overlaps with the emission spectrum of the first reporter group; or (ii) an emission spectrum that overlaps with the excitation spectrum of the first reporter group.
The selected first protein (e.g., the amino acid sequence thereof) may be novel or known. However, in many instances, the function of the first protein will not be known. In a non-limiting example, identifying a protein not previously known to have ligand binding activity may comprise a structurally assisted functional evaluation (SAFE) homolog search method comprising the following steps:
(1) Collecting a sequence homology set using a BLAST sequence alignment tool starting with ligand binding protein sequence disclosed herein as a seed.
Permissive settings are used, such that pairwise hits are required to have a minimum of only, e.g., 20%, 25%, 30%, 35% or 40% sequence identity with the seed sequence. The lengths of the hit and seed are mutually constrained such that the alignment covers at least, e.g., 60%, 65%, 70%, 85%, or 90% within each partner.
(2) Structure-based encoding of biological function: A primary complementary surface (PCS) comprising the protein residues that form hydrogen bonds and van der Waals contacts with a bound ligand is defined using computer-assisted, visual inspection of the three-dimensional structure of the biosensor-ligand complex. This definition specifies residue positions and their permitted amino acid identity. Multiple amino acid identities are permitted at each position to encode functionally equivalent residues. This definition establishes a search filter for the accurate prediction of ligand-binding proteins within the universe of sequence homologs collected in (1).
(3) Accurate sequence alignment: Tools such as ClustalW are used to construct an accurate alignment of all the sequence homologs. The ligand-binding protein seed sequence is included in the alignment. This multiple sequence alignment establishes the equivalent positions of the seed sequence (primary complementary surface) PCS in each sequence homolog.
(4) Function evaluation: The ligand-binding properties of each of the aligned sequence homologs is determined by measuring their compliance with the PCS
sequence filter. A "Hamming distance", H, is assigned for each homolog, which specifies the degree of sequence identity of all the residues at the aligned PCS positions. A value of H=0 indicates that the identities of all the residues at the aligned PCS positions match the amino acid(s) allowed in the PCS search filter; H>0, indicates that one or more aligned positions have disallowed residues. Sequences for which H=0 are predicted to encode ligand-binding proteins.
(5) Selection of representative SAFE homologs: The sequence homologs are ordered by (a) identity with the seed PCS, as measured by the Hamming distance, (b) fractional overall sequence identity with the seed sequence. A subset for sequences with H=0, sampling the fractional overall sequence identity is selected for experimental verification.
In a non-limiting example, identifying a protein not previously known to have ligand binding activity may comprises the following steps:
(1) performing a computational search of sequence databases to define a broad group of simple sequence or structural homologs of any known, ligand binding protein;
(2) using the list from step (1), deriving a search profile containing common sequence and/or structural motifs shared by the members of the list [e.g. by using computer programs such as MEME (Multiple Em for Motif Elicitation available at meme.sdsc.edu/meme/cgi-bin/meme.cgi) or BLAST];
(3) searching sequence/structural databases, using a derived search profile based on the common sequence or structural motif from step (2) as query (e.g., using computer programs such as BLAST, or MAST (Motif Alignment Search Tool available at meme.sdsc.edu/meme/cgi-bin/mast.cgi), and identifying a candidate sequence, wherein a sequence homology and/or structural similarity to a reference ligand binding protein is a predetermined percentage threshold;
(4) compiling a list of candidate sequences to generate a list of candidate ligand binding proteins;
(5) expressing the candidate ligand-binding proteins in a host organism; and (6) testing for ligand binding activity, wherein detection of ligand binding in the organism (or the media thereof) indicates that the candidate sequence comprises a novel ligand binding protein.
In non-limiting examples, the MEME suite of sequence analysis tools (meme.sdsc.edu/meme/cgi-bin/meme.cgi) can also be used as an alternative to BLAST.
Sequence motifs are discovered using the program "MEME". These motifs can then be used to search sequence databases using the program "MAST." The BLAST search algorithm is well-known.
In various embodiments relating to alignments using a ClustalW aligment program, the ClustalW alignment program may be, e.g., ClustalW alignment program version 2.1.
Each embodiment disclosed herein is contemplated as being applicable to each of the other disclosed embodiments. Thus, all combinations of the various elements described herein are within the scope of the invention.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below.

DESCRIPTION OF THE DRAWINGS
FIGS. lA and B are cartoons showing fluorescent probes. FIG. 1A is a cartoon relating to indirect fluorescent responses. Fluorescent biosensors can be constructed by site-specifically attaching a fluorophore to a protein that undergoes a conformational change upon binding ligand (triangle) in a location between the two lobes of the protein (periplasmic binding protein or engineered derivative thereof), such that the shape and intensities of the fluorescent conjugate emission spectra changes. FIG. 1B is a cartoon relating to direct fluorescent responses. Fluorescent chemosensors based on fluorophores that interact directly with an analyte.
FIGS. 2A-C are graphs illustrating ratiometry. If the fluorescence emission spectrum changes shape in response to binding of an analyte, such as glucose, then the ratio of emission intensities at two appropriately chosen wavelengths reports on analyte concentration. FIG. 2A: In the absence of ligand, the emitted fluorescence color is predominantly blue, whereas the ligand complex fluoresces green. Arrows indicate the direction of change upon ligand addition. FIG. 2B: The ligand dependence of the absolute blue and green intensities. FIG. 2C: The ratio of the blue and green intensities reports enables ligand binding to be determined.
FIGS. 3A-D are graphs and diagrams showing three dominant factors that affect overall ligand-mediated change in fluorescence emission intensity between donor and acceptors in which one partner responds to ligand binding. FIG. 3A: Simplified Jablonski diagram illustrating radiative and non-radiative pathways in the donor and acceptor. The donor excited state (D*) is formed through illumination by the excitation source (wavy arrow) whereas the acceptor excited state (A*) is formed by resonance energy transfer (dashed arrow). The fluorescence intensity is determined by the ratio of radiative decay (gray arrows) of the excited states (gray lines) to the ground state (black line) relative to all non-radiative processes (black arrows), and the resonance energy transfer rate, kt, from donor to acceptor. FIG. 3B: Inter-dipole geometry. Top, energy transfer efficiency ( f = Qr I (Q0 ¨ Q,), where the Qr, Qo, Q , are the quantum efficiencies at distances r, closest approach, and infinity, respectively) varies as the 6th power of the distance between two dipoles. Bottom, energy transfer efficiency varies as the square of the orientation factor K, where K = sin D sin Acos x ¨ 2 cos OD cos OA with OD and OA the angles of the donor (blue) and acceptor (red) electronic transition dipoles with the line connecting them, and x the angle between the planes within which they lie. FIG. 3C: Spectral overlap (gray area) between the donor fluorescence emission (DJ, blue) and acceptor fluorescence excitation (AA, black) spectra. This overlap increases with bathochromic or hypsochromic shifts of the donor emission (red arrow) and acceptor excitation (dotted blue arrow) spectra, respectively. Shifts in the opposite directions decreases spectral overlap.
FIGS. 4A-C are illustrations of the construction of ngmFRET pairs by combining a fluorophore that responds directly to ligand binding with a non-interacting, indirectly responsive partner. FIG. 4A: A glucose-binding protein in which a directly responsive partner is positioned in the vicinity of the glucose-binding site, and the indirectly responsive partner is fused as the protein C-terminus. FIG. 4B: The internally positioned fluorophore of Yellow Fluorescent Protein responds directly chloride binding by itself and regardless of the presence of any other fluorophore/partner. Its monochromatic response can be converted to a dichromatic one by positioning a second, indirectly responsive fluorophore on the surface of the protein. FIG. 4C: An adaptor protein such as E. coli thioredoxin can be used to position an directly responsive chemosensor next to an indirectly responsive partner, thereby converting a monochromatic into a dichromatic signal.
FIGS. 5A-E are cartoons of fusion constructs that enable site-specific labeling of cysteines at two independently addressable sites with distinct, thiol-reactive fluorophores.
FIG. 5A: In the first labeling step, an unprotected single thiol (circle) reacts with a fluorophore, while thiols at a second site remain protected within a disulfide bridge. In the second labeling step, the disulfide is deprotected by reduction, and fluorophores are coupled to the second site. FIG. 5B: C-terminal fusion of a PZif domain (slanted lines) to ttGGBP
(solid gray). FIG. 5C: N-terminal fusion of PZif. FIG. 5D: C-terminal fusion ecTRX
(horizontal lines). FIG. 5E: N-terminal fusion of ecTRX.
FIG. 6 is a structural depiction of ionization states affecting fluorescence of the YFP
fluorophore (by itself and regardless of the presence of any other fluorophore/partner).
FIG. 7 shows the sequence of laYFP and the locations of cysteine mutations for the construction of semisynthetic chloride sensors. Gray: the tripeptide that forms the fluorophore in the matured protein. Underlined: mutations that tune the YFP
wavelength relative to GFP. laYFP retains the wild-type GFP residues H148 and V68 which affect chloride location and affinity (Wang 2015). Standard numbering is used in which the start methionine is 0. Structure taken from Protein Data Bank (PDB) Accession Code:
3sve.

FIGS. 8A and B are structures showing the locations of the cysteine mutations on the surface of laYFP. Arrows indicate (3-barrel; central a helix; fluorophore; and chloride-binding site (dark gray sphere). Light gray spheres indicate location of surface cysteine mutations (numbered according to Fig. 7). FIG. 8A: Side-view showing that the cysteine mutations on the surface of the barrel (3 strands form an annulus that approximately encircles the fluorophore in the hydrophobic core. FIG. 8B: Top view showing the positions of all the annulus cysteine mutations around the barrel (end mutations omitted for clarity).
FIGS. 9A and B are graphs showing the chloride-dependent responses of the emission intensity spectra of representative Acrylodan and Pacific Blue YFP conjugates.
Left column, normalized corrected emission spectra (see notes to Table 2): purple line, no chloride; red line, high chloride concentration; thin black lines, intermediate concentrations. YFP emission intensity peak is centered at 530 nm in both conjugates. Arrows indicate direction of change with increased chloride concentrations. Middle column, fit of ratiometric signal (R12) to a Langmuir binding isotherm (yields aPPKd; see notes to Table 2). Right column:
fits of Langmuir binding isotherm to monochromatic intensity (/) signals (yields mieKd); gray circles, YFP intensity; black circles, Acrylodan or Pacific Blue intensity. FIG. 9A:
C1BP4=Acrylodan (k1 = 530 nm, k2= 500 nm; aPPKd = 41 mM; mieKd=129 mM). FIG. 9B:
C1BP10=Pacific Blue (Xi = 530 nm, k2= 455 nm; aPPKd = 260 mM; frueKd=105 mM).
FIG. 10 is an illustration of a structure showing positions for introducing cysteine mutations in ttGGBP to which fluorophores can be covalently coupled for reagentless biosensor construction. Positions 17, 91, 151, and 182 are endosteric;
positions 11, 16, 42, 67, 92, 111, 148, 152, 181, and 183 are peristeric; and positions 257, 259, and 300 are allosteric FIGS. 11A-P are illustrations of fluorophore structures. Naphthalene family (arrows indicate known or potential internal twists): FIG. 11A shows Acrylodan; FIG.
11B shows Badan; FIG. 11C shows IAEDANS. Xanthene family: FIG. 11D shows Fluorescein (5-IAF
and 6-IAF); FIG. 11E shows Oregon Green; FIG. 11F shows Alexa 432; FIG. 11G
shows Alexa 532; FIG. 11H shows Alexa 546; FIG. 11I shows Texas Red. Coumarin family: FIG.
11J shows Pacific Blue; FIG. 11K shows CPM. benzoxadiazole family: FIG. 11L
shows IANBD. Boradiazaindacine (BODIPY) family: FIG. 11M shows BODIPY 499/508; FIG.
11N shows BODIPY 507/545. Cyanine family: FIG. 110 shows Cy5. Miscellaneous:
FIG.
11P shows PyMPO.

FIGS. 12A and B is a pair of graphs showing donor quenching effects. FIG. 12A:

The normalized fluorescence intensity (1(2); purple: apo-protein; red, high glucose; thin black line: intermediate concentrations) of the singly labeled F 1 7C=Pacific Blue conjugate increases (blue arrow) in response to glucose binding without significant shifts in the wavelength of the intensity maximum. FIG. 12B: In the doubly labeled fusion protein, the fluorescence emission intensities (color scheme as in FIG. 12A) of the Fl7C=Pacific Blue directly responsive donor and the f3ZiPIAF indirectly responsive acceptor both increase in response to glucose binding. In the absence changes in spectral overlap, the observed intensity response pattern is consistent with decreases in the non-radiative decay rate of the Pacific Blue donor (model crgf, Table 1). The relative changes in donor and acceptor emissions are unequal, enabling ratiometric sensing (inset) based on the ratio (R12) of intensities at 456 nm (donor) and 520 nm (acceptor) so that the glucose concentration can be fit to a single-site Langmuir binding isotherm (equations 22 and 24); constant baselines for the apo-protein and ligand complex; aPPKd = 31 mM).
FIG. 13A is a cartoon and FIG. 13B is a structural illustration relating to ratiometric sensing using Forster resonance energy transfer between pairs of glucose-responsive and non-responsive fluorophores attached site-specifically in a fusion protein that enables orthogonal site-specific cysteine labeling. FIG. 13A: Fusion of a single cysteine ttGGBP
mutant (gray line; two alternative cysteine positions indicated, i.e., F 17C and W182C) and a disulfide-containing PZif domain (disulfide indicated), separated by a linker (thin line), enables sequential labeling with two different fluorophores (line sizes indicate relative size of the fusion domains). FIG. 13B: Model showing the positions of the C-terminal PZif fusion relative to the experimentally determined F17C=Badan and W182C=Acrylodan conjugates of ttGGBP. This domain is connected to ttGGBP via a flexible linker, and is therefore likely to adopt an ensemble of conformations, the approximate extent which is indicated by the oval.
FIGS. 14A-D are graphs showing acceptor dipole switching and quenching effects in fluorescein conjugates. FIG. 14A: W182C=5-IAF (directly responsive acceptor) PZif=Pacific Blue (indirectly responsive donor). Normalized emission intensities are colored according to glucose concentration range: purple line, apo-protein; dotted gray line, emission at saturated glucose level for higher affinity binding site (phase I response, see main text); red line, emission intensity at highest glucose concentration measured; solid black, intermediate glucose concentrations for phase I; dotted black lines, elevated glucose concentrations in the phase II response. Directions of signal change: bottom arrow, phase I; top arrow, phase II.
Inset, Langmuir binding isotherm of ratiometic signal R 1 2 at 456 nm and 520 nm, aPPKd = 2.9 mM, constant and linear baselines for apo-protein and ligand complex respectively. FIG.
14B: Contour plot of FIG. 14A, indicating phase I and phase II responses (see main text).
FIG. 14C: W182C=Oregon Green (directly responsive acceptor) gif=Pacific Blue (indirectly responsive donor) coloring as in FIG. 14A. FIG. 14D: Contour plot of FIG. 14C.
FIGS. 15A and B are graphs showing glucose-dependent emission spectra of Fl7C=Badan and W182C=Acrylodan conjugates of ttGGBP. Corrected spectra (apo-protein, dark red; saturated glucose, purple; intermediate glucose concentrations, black). Insets, fit of the ratiometric signal (equation 1 and 2; 20 nm integration bandwidth): gray circles, experimentally observed ratios; black line, calculated fit (baselines; apo-protein, constant;
saturated glucose complex, linear). FIG. 15A: Fl7C=Badan (hypsochromic; ki, 470 nm; k2, 542 nm; aPPKd, 0.18 mM); FIG. 15B: F182C=Acrylodan (bathochromic; Xi, 475 nm;
k2, 545 nm; aPPKd, 2.2 mM).
FIGS. 16A-D are graphs showing glucose dependence of electronic transitions in the fluorescence emission intensity spectra of Fl7C=Badan and W182C=Acrylodan ttGGBP
conjugates. Columns: left, singular value decomposition (SVD) of the glucose-dependent corrected spectra; right, Gaussian analysis of electronic transitions in the fluorescence emission intensity. FIGS. 16A and B: F17C=Badan; FIGS. 16C and D:
F182C=Acry1odan.
For SVD analysis, frequency transformations of the spectra (equation 30) were decomposed into principal components (equation 31). The contribution of the first component, Ci (black), is largely invariant with glucose concentration, whereas the second component, C2 (red), encode accounts for the glucose-dependent changes in the spectra (inserts:
glucose dependence of the fractional contribution of each component, equation 32). In the Gaussian analysis, the spectral emission intensities can be accounted for to a first approximation by two excited state electronic transitions: a low-energy, Si (Acrylodan: 521 9 nm; Badan:
530 14 nm) and a high-energy, S2 (Acrylodan: 477 14 nm; Badan: 477 16 nm) transition.
Glucose binding shifts the population of these excited states. At each titration point, the experimentally observed emission intensities were modeled with Gaussians fits (equation 33) for Si and S2 electronic transition. Experimental emission spectra and Gaussian fits are shown only for the apo-protein, and saturated glucose complex. Emission spectra: dashed black line, apo-protein; solid black line, glucose complex. Gaussians (Si, lines; 52, lines):
dashed lines, apo-protein; solid lines, glucose complex. Thin black lines:
residuals (equation 35) at each titration point. Inserts show the population fractions (equation 34) of the Si and S2 transitions extracted from the spectra at each titration point (black circles) fit to Langmuir binding isotherms (solid lines) with aPPKd values constrained to be the same for both populations. As a first approximation, the wavelengths of Si or S2 transitions are the same in apo-protein and the saturated glucose complex. The residuals indicate that a more extensive treatment is required in which the Si and S2 are split into multiple transitions to fully fit the spectra. Wavelength shifts occur if there is a significant redistribution of the two excited state populations in the apo-protein and the saturated ligand complexes. In the bathochromic ratiometric 182C=Acrylodan conjugate, the Si state dominates in the glucose complex (FIG.
16D); in the hypsochromic conjugate 17C=Badan (FIG. 16B) the apo-protein comprises a mixture of the two states, whereas the glucose complex contains almost exclusively the S2 state.
FIGS. 17A-D are graphs showing glucose dependence of the absorption spectra of ttGGBP Acrylodan and Badan conjugates that undergo wavelength shifts in their fluorescence emission intensities in response to ligand binding Columns: left, singular value decomposition (SVD) of the glucose-dependent corrected spectra; right, Gaussian analysis of electronic transitions in the absorption spectra. FIGS. 17A and B: F17C=Badan;
FIGS. 17C
and D: F182C=Acrylodan. For SVD analysis, frequency transformations of the spectra (equation 30) were decomposed into principal components (equation 31). Inserts show glucose dependence of the fractional contribution of each component (equation 32). As with the fluorescence emission spectra (FIG. 16), in the Gaussian analysis, the spectral emission intensities can be accounted for to a first approximation by two ground state electronic transitions (dashed black line, apo-protein; solid black line, glucose complex): Gi (386 nm) and G2 (359 nm; apo-protein, dashed; glucose complex, solid). Inset, glucose dependence of population fractions at each glucose titration (black circles) of the Gi and G2 transitions fit to Langmuir binding isotherms (solid lines) with aPPKd values constrained to be the same for both populations (residuals, thin lines).
FIGS. 18A-F are a set of graphs showing donor dipole switching effects in Acrylodan and Badan conjugates. Four doubly labeled conjugates were constructed, in which Acrylodan or Badan directly responsive donors were combined with A1exa532 or 5-IAF
indirectly responsive acceptors. FIG. 18A: F17C=Badan gif Alexa532. 1(2) , normalized emission intensity: purple line, apo-protein; red line, saturating glucose concentration; thin black lines, intermediate glucose concentrations. Blue arrow, direction of change with increased glucose concentration. Inset, Langmuir binding isotherm of ratio, R12, of the intensities at 467 nm and 560 nm (equations 23 and 24; constant baselines for apo-protein and ligand complex;
aPPKd = 0.16 mM). FIG. 18B: F17C=Badan f3Zif5-IAF (color scheme as for FIG.
18A). Inset, Langmuir isotherm of ratiometric signal R12 at 467 nm and 520 nm, aPPKd = 0.18 mM, constant and linear baselines for apo-protein and ligand complex respectively.
FIG. 18C:
W182C=Acrylodan PZifAlexa532. Normalized emission intensities are colored according to glucose concentration range: purple line, apo-protein; dotted red line, emission at saturated glucose level for higher affinity binding site (phase I response, see main text); red line, emission intensity at highest glucose concentration measured; solid black, intermediate glucose concentrations for phase I; dotted black lines, elevated glucose concentrations in the phase II response. Directions of signal change: blue arrow, phase I; red arrow, phase II.
Inset, Langmuir binding isotherm of ratiometric signal R12 at 480 nm and 550 nm, aPPKd = 1.7 mM, constant and linear baselines for apo-protein and ligand complex respectively. FIG.
18D: W182C=Acrylodan f3Zif5-IAF. Coloring according to FIG. 18C. Inset, Langmuir binding isotherm of ratiometric signal Ri2 at 465 nm and 520 nm, aPPKd = 1.9 mM, constant and linear baselines for apo-protein and ligand complex respectively. FIG.
18E:
W182C=Acrylodan PZifAlexa532 contour plot of the glucose dependence of emission intensities, indicating phase I and phase II responses (see main text). FIG.
18F:
W182C=Acry1odan f3Zif5-IAF contour plot.
FIGS. 19A and B are graphs providing a comparison of singly-labeled ttGGBP17C=Badan (A) and the C-terminal doubly labeled ttGGBP17C=Badan-ecTRX=Alexa532 fusion (B). Note the appearance of the approximately invariant emission peak (arrow) of the indirectly responsive A1exa532 acceptor.
FIGS. 20A-D are graphs showing donor dipole switching effects in W182CIAEDANS. FIG. 20A: Singular value decomposition analysis of the change in the emission intensity of singly labeled W182CIAEDANS in response to glucose, showing the wavenumber dependence of the invariant (black line) and variant (red line) spectral components. Inset shows change in contribution of the two spectral components with respect to glucose concentration. FIG. 20B: The glucose response is accounted for largely by two electronic transitions (green, 542 nm; blue, 485 nm) which were fits as Gaussians to the experimental emission intensities (purple line, in the absence of glucose; red line, saturating glucose). Black lines show residuals between observed and calculated spectra.
Inset shows 1o the change in the populations of the blue and green transitions in response to glucose. FIG.
20C: W182C=IAEDANS f3Zif5-IAF. Normalized emission intensities, I(2), are colored according to glucose concentration range: purple line, apo-protein; dotted red line, emission at saturated glucose level for higher affinity binding site (phase I response, see main text); red line, emission intensity at highest glucose concentration measured; solid black, intermediate glucose concentrations for phase I; dotted black lines, elevated glucose concentrations in the phase II response. Directions of signal change: blue arrow, phase I; red arrow, phase II.
Inset, Langmuir binding isotherm of ratiometic signal R12 at 465 nm and 520 nm, aPPKd =
0.09 mM, constant and linear baselines for apo-protein and ligand complex respectively.
FIG. 20D: Contour plot of the glucose dependence of W182CIAEDANS f3Zif5-IAF
emission intensities, indicating phase I and phase II responses (see main text).
FIG. 21 is a structural illustration of ionization equilibria of the fluorescein carboxylate and phenolic hydroxyl (Martin 1975).
FIG. 22 is an illustration of the structure of E. coli thioredoxin (Kati 1990) showing positions of mutations constructed in the adaptor proteins. Disulfide (C32,C35) is indicated.
The D2A, D26A, and K57M background mutations constructed in all adaptor proteins are indicated (D2A removes adventitious N-terminal Cu(II)-binding site; D26A and remove charges buried in the hydrophobic core). Large gray spheres: surface lysines mutated to arginine in Adaptor2.0a (K4Q and K1 8Q in Adaptor2.0b). Structure from PDB
accession code 2trx.
FIG. 23 shows amino acid sequences of the engineered adaptor proteins based on E.
coli thioredoxin. Numbering according the X-ray structure of the mature protein, lacking the initial methionine. Wild-type sequence is shown in full, mutations are given below (blank indicates wild-type residue).
FIGS. 24A-D are graphs showing the pH dependence of the emission spectra of Adaptor1.0 conjugates. Left column: corrected fluorescence emission intensity spectra (see notes to Table 9) of doubly labeled conjugates (purple, pH 4.0; red, pH 9.5;
thin black lines, intermediate values at 0.5 pH unit intervals); middle column: ratiometic response, R12 = 11/12 (black circles), as a function of pH (gray lines, fits to Langmuir binding isotherm give aPPpKa values), where /1 and 12 are the intensities integrated over a 20 nm band centered at k1 and k2;
right column: integrated fluorescence emission intensity changes, I, at the two individual wavelengths as a function of pH (gray lines, fits give mlepKa values). In all cases /1 (gray circles) corresponds to the Fluorescein integrated intensities at 520 nm (red increases with pH, arrows), and 12 (black circles) to 460 nm and 455 nm for Acrylodan and Pacific Blue (PB), respectively. FIG. 24A: Fluorescein attached to C73, and Acrylodan to the disulfide (aPPpKa = 6.22, mlepKa = 6.16). FIG. 24B: Fluorescein attached to the disulfide, and Acrylodan to C73 (aPPpKa = 6.09, mlepKa = 6.13). FIG. 24C: Fluorescein attached to C73, Pacific Blue to the disulfide (aPPpKa = 6.26, tuepKa = 6.22); FIG. 24D:
Fluorescein attached to the disulfide, Pacific Blue to C73 (aPPpKa = 6.14, thlepKa = 6.17).
FIGS. 25A-D are graphs showing the pH dependence of the absorption spectra of Adoptor1.0 conjugates. Left column: corrected absorbance, A(20, spectra (see notes to Table 9) of doubly labeled conjugates (purple, pH 4.0; red, pH 9.5; thin black lines, intermediate values at 0.5 pH unit intervals); middle column: ratiometric response, R12 =
Ail A2 (black circles), as a function of pH (gray lines, fits to Langmuir binding isotherm give aPPpKa values), where A1 and A2 are the absorbances integrated over a 20 nm band centered at Xi and X2; right column: integrated absorbance changes, A, at the two individual wavelengths as a function of pH (gray lines, fits give mlepKa values). In all cases A1 (gray circles) corresponds to the Fluorescein integrated absorbances at 495 nm (increases with pH, arrows), and A2 (black circles) to 390 nm and 410 nm for Acrylodan and Pacific Blue (PB), respectively (decreases with pH, arrows). FIG. 25A: Fluorescein attached to C73, and Acrylodan to the disulfide (aPPpKa = 6.52, mlepKa = 6.40). FIG. 25B: Fluorescein attached to the disulfide, and Acrylodan to C73 (aPPpKa = 6.70, mlepKa = 6.62). FIG. 25C: Fluorescein attached to C73, Pacific Blue to the disulfide (aPPpKa = 6.72, tuepKa = 6.77); FIG. 25D:
Fluorescein attached to the disulfide, Pacific Blue to C73 (aPPpKa = 6.73, thlepKa = 6.66).
FIGS. 26A and B are graphs showing the pH dependence of the fluorescence emission and absorption spectra of Adaptor2.0a conjugate labeled with Fluorescein at the amino terminus, and Acrylodan at the disulfide. Left column, corrected spectra (purple, pH 4.0; red, pH 9.5; thin black lines, intermediate values at 0.5 pH unit intervals).
Middle column:
ratiometric responses (black circles, ratiometric signals; gray lines, fits to Langmuir binding isotherm give aPPpKa values). Right column, integrated changes at the two individual wavelengths gray circles, k1; black circles, k2) as a function of pH (gray lines, fits give fruepKa values). FIG. 26A: pH dependence of fluorescence emission (Xi = 520 nm, k2 =
460 nm;
aPPpKa = 5.83, mlepKa = 5.82). FIG. 26B: pH dependence of absorption properties (Xi = 498 nm, k2 = 375 nm; aPPpKa = 6.36, mlepKa = 6.08).

FIGS. 27A-F are graphs showing temperature dependence and pH dependence of Adaptor1.0 and Adaptor 2.0a fluorescent conjugates measured on a Roche LightCycler. Left column: Adaptor1.0 R73C=Pacific Blue, disulfide=Fluorescein, fluorescence ratio recorded at 488 nm and 510 nm; right column: Adaptor 2.0=Fluorescein, disulfide Pacific Blue, fluorescent ratio recorded at 488 nm and 580 nm. FIGS. 27A, B: pH- and temperature-dependent landscape of the fluorescence ratio (Z axis): dashed-dotted line, approximate mid-point concentrations (pKa) of the response to pH; dashed line, approximate mid-point temperatures (Tin) of thermal stability (equation 36). FIGS. 27C, D: Thermal melt at pH 4.5, as monitored by R12=/(488 nm)//(510 nm) emission intensity ratio: blue, experimental data;
green, fit to two-state van 't Hoff thermal denaturation (T. ¨360K). FIGS.
27E, F:
Temperature dependence of pKa values: crosses, measured pKa values; line, fit to Gibbs-Helmholtz temperature dependence of the free energy all+ binding (equation 37).
FIG. 28 is a diagram relating to directly responsive partners and indirectly responsive partners in ngmFRET pathways.
FIG. 29 shows the sequence of an exemplary chloride-binding protein (YFP) 1 (C1BP1) expression construct (SEQ ID NO: 46).
FIG. 30 shows the sequence of an exemplary C1BP2 expression construct (SEQ ID
NO: 47).
FIG. 31 shows the sequence of an exemplary C1BP3 expression construct (SEQ ID
NO: 48).
FIG. 32 shows the sequence of an exemplary C1BP4 expression construct (SEQ ID
NO: 49).
FIG. 33 shows the sequence of an exemplary C1BP5 expression construct (SEQ ID
NO: 50).
FIG. 34 shows the sequence of an exemplary C1BP6 expression construct (SEQ ID
NO: 51).
FIG. 35 shows the sequence of an exemplary C1BP7 expression construct (SEQ ID
NO: 52).
FIG. 36 shows the sequence of an exemplary C1BP8 expression construct (SEQ ID
NO: 53).
FIG. 37 shows the sequence of an exemplary C1BP9 expression construct (SEQ ID
NO: 54).

FIG. 38 shows the sequence of an exemplary C1BP10 expression construct (SEQ ID

NO: 55).
FIG. 39 shows the sequence of an exemplary C1BP11 expression construct (SEQ ID

NO: 56).
FIG. 40 shows the sequence of an exemplary C1BP12 expression construct (SEQ ID
NO: 57).
FIG. 41 shows the sequence of an exemplary C1BP13 expression construct (SEQ ID

NO: 58).
FIG. 42 shows the sequence of an exemplary C1BP14 expression construct (SEQ ID
NO: 59).
FIG. 43 shows the sequence of an exemplary ttGGBP.11C.O.bZif expression construct (SEQ ID NO: 60).
FIG. 44 shows the sequence of an exemplary ttGGBP.17CØbZif expression construct (SEQ ID NO: 61).
FIG. 45 shows the sequence of an exemplary ttGGBP.111C.O.bZif expression construct (SEQ ID NO: 62).
FIG. 46 shows the sequence of an exemplary ttGGBP.151CØbZif expression construct (SEQ ID NO: 63).
FIG. 47 shows the sequence of an exemplary ttGGBP.182C.O.bZif expression construct (SEQ ID NO: 64).
FIG. 48 shows the sequence of an exemplary ttGGBP.17C.3.Trx expression construct (SEQ ID NO: 65).
FIG. 49 shows the sequence of an exemplary Trx.ttGGBP.17C.3 expression construct (SEQ ID NO: 66).
FIG. 50 shows the sequence of an exemplary ttGGBP.182C.2.Trx expression construct (SEQ ID NO: 67).
FIG. 51 shows the sequence of an exemplary Trx.ttGGBP.182C.2 expression construct (SEQ ID NO: 68).
FIG. 52 shows the sequence of an exemplary Adaptor expression construct (SEQ
ID
NO: 69).
FIG. 53 shows the sequence of an exemplary Adaptor1.0 expression construct (SEQ
ID NO: 70).

FIG. 54 shows the sequence of an exemplary Adaptor2.0a expression construct (SEQ
ID NO: 71).
FIG. 55 shows the sequence of an exemplary Adaptor2.0b expression construct (SEQ
ID NO: 72).
FIG. 56 shows the sequence of an exemplary Adaptor3.0 expression construct (SEQ
ID NO: 73).
FIG. 57 shows the sequence of an exemplary Adaptor4.0 expression construct (SEQ
ID NO: 74).
FIG. 58 shows the sequence of an exemplary Adaptor5.0 expression construct (SEQ
lD NO: 75).
FIG. 59 shows the sequence of an exemplary Adaptor6.0 expression construct (SEQ
1D NO: 76).
FIG. 60 shows the sequence of an exemplary Adaptor7.0 expression construct (SEQ
1D NO: 77).
FIG. 61 shows the sequence of an exemplary Adaptor8.0 expression construct (SEQ
1D NO: 78).
FIG. 62 shows the sequence of an exemplary Adaptor9.0 expression construct (SEQ
1D NO: 79).
FIG. 63 shows the sequence of an exemplary Adaptorl 0.0 expression construct (SEQ
lD NO: 80).
FIG. 64 shows the sequence of an exemplary Adaptorl 1.0 expression construct (SEQ
1D NO: 81).
FIG. 65 shows the sequence of an exemplary Adaptorl 2.0 expression construct (SEQ
1D NO: 82).
FIG. 66 shows the sequence of an exemplary Adaptor13.0 expression construct (SEQ
1D NO: 83).
FIG. 67 shows the sequence of an exemplary Adaptor14.0 expression construct (SEQ
1D NO: 84).
FIG. 68 shows the sequence of an exemplary Adaptorl 5.0 expression construct (SEQ
lD NO: 85).
FIG. 69 shows the sequence of an exemplary Adaptorl 6.0 expression construct (SEQ
1D NO: 86).

DETAILED DESCRIPTION
Biosensors are analytical tools that can be used to measure the presence of a single molecular species in a complex mixture by combining the exquisite molecular recognition properties of biological macromolecules with signal transduction mechanisms that couple ligand binding to readily detectable physical changes (Hall, Biosensors, Prentice-Hall, Englewood Cliffs, N.J.; Scheller et al., Curr. Op. Biotech. 12:35-40, 2001).
Ideally, a biosensor is reagentless and, in contrast to enzyme-based assays or competitive immunoassays, does not change composition as a consequence of making the measurement (Hellinga & Marvin, Trends Biotech. 16:183-189, 1998). Most biosensors combine a naturally occurring macromolecule such as an enzyme or an antibody, with the identification of a suitable physical signal particular to the molecule in question, and the construction of a detector specific to that system (Meadows, Adv. Drug Deliv. Rev. 21:177-189, 1996).
Recently, molecular engineering techniques have been explored to develop macromolecules that combine a wide range of binding specificities and affinities with a common signal transduction mechanism, to construct a generic detection system for many different analytes (Hellinga & Marvin, Trends Biotech. 16:183-189, 1998).
Escherichia coli periplasmic binding proteins are members of a protein superfamily (bacterial periplasmic binding proteins, bPBPs) (Tam & Saier, Microbiol. Rev.
57:320-346, 1993). These proteins comprise two domains linked by a hinge region (Quiocho &
Ledvina, Molec. Microbiol. 20:17-25, 1996). The ligand-binding site is located at the interface between the two domains. The proteins typically adopt two conformations: a ligand-free open form, and a ligand-bound closed form, which interconvert via a hinge-bending mechanism upon ligand binding. This global, ligand-mediated conformational change has been exploited to couple ligand binding to changes in fluorescence intensity by positioning single, environmentally sensitive fluorophores in locations that undergo local conformational changes in concert with the global change (Brune et al., Biochemistry 33:8262-8271, 1994;
Gilardi et al., Prot. Eng. 10:479-486, 1997; Gilardi et al., Anal. Chem.
66:3840-3847, 1994;
Marvin et al., Proc. Natl. Acad. Sci. USA 94:4366-4371, 1997, Marvin and Hellinga, J. Am.
Chem. Soc. 120:7-11, 1998; Tolosa et al., Anal. Biochem. 267:114-120, 1999;
Dattelbaum &
Lakowicz, Anal. Biochem. 291:89-95, 2001; Marvin & Hellinga, Proc. Natl. Acad.
Sci. USA
98:4955-4960, 2001; Salins et al., Anal. Biochem. 294:19-26, 2001).
Provided herein are improved biosensors that rapidly, reliably, and accurately detect and quantify ligands with significant advantages over previous systems. The present disclosure provides a biosensor for ligand, comprising a ligand-binding protein that is attached to one or more reporter group (e.g., 1, 2, 3, or more reporter groups). The binding of a ligand to the ligand-binding domain of the ligand-binding protein causes a change in signaling by the biosensor. In various implementations, the biosensor may produce a signal when a ligand is bound to the ligand binding domain that is not produced (and/or that is different from a signal that is produced) when the ligand is absent from the ligand binding domain. These biosensors have widespread utility including in clinical, industrial, and environmental settings.
Various biosensors provided herein produce a dichromatic, ratiometric signal, i.e., the signal is defined as the quotient of the intensities at two independent wavelengths. The advantage of such a signal is that it provides an internally consistent reference. The self-calibrating nature of a ratiometric measurement removes the necessity for carrying out on-board calibration tests prior to each measurement. The biosensors are reagentless in that their monitoring mechanism requires neither an enzyme nor additional substrates for a signal to develop, nor measurement of substrate consumption or product generation rates to determine ligand concentrations.
Reagentless, fluorescently responsive biosensors present a number of advantages over enzyme-based biosensors, including elimination of chemical transformations, elimination of substrate requirements, and self-calibration, which together lead to rapid response times, continuous monitoring capabilities, simple sample-handling, and lower cost due to simplified manufacturing and distribution processes.
ngmFRET for Ratiometric Measurements Using Reagentless Analyte Sensors Determination of analyte concentrations using fluorescent probes is a powerful technique in analytical chemistry (FIGS. lA and B). Fluorescent chemosensors based on small-molecule fluorophores that interact directly with an analyte (Zhang, Yin and Yoon 2014, Lavis and Raines 2008, Lavis and Raines 2014) and fluorescent biosensors based on engineered proteins that couple analyte-binding events to changes in the emission properties of fluorophores (being fluorescent by themselves and regardless of the presence of any other fluorophore/partner) (Okumoto 2012) or semi-synthetically (Wang 2009) incorporated fluorophores have wide-ranging applications in cell biology and analytical chemistry(Borisov and Wolfbeis 2008, Liu 2015, Matzeu 2015, Heo and Takeuchi 2013). If the fluorescence emission spectrum changes shape in response to analyte binding such that the ratio of emission intensities at two appropriately chosen wavelengths reports on analyte concentration (dichromatic response), then ratiometric measurements can be used to monitor analyte concentrations (FIGS. 2A-C). Ratiometry is essential for devices that rely on quantifying changes in fluorescence emission intensities, because it provides an internally consistent reference (Demchenko 2010, Demchenko 2014). The self-calibrating nature of a ratiometric measurement removes the necessity for carrying out on-board calibration tests prior to each measurement (Choleau et al. 2002), obviating the need for multiple components and fluidic circuitry. Accordingly, reagentless, ratiometric fluorescent sensors have many uses in process engineering, environmental or clinical chemistry, including single-use point-of-care applications (Kozma et al. 2013, Ahmed et al. 2014, Mohammed 2011, Ispas 2012, Rogers and Boutelle 2013, Robinson and Dittrich 2013, Arora et al. 2010, Gubala et al. 2012), wearable devices (Badugu, Lakowicz and Geddes 2005), optodes for continuous monitoring (Weidemaier et al. 2011, Judge et al. 2011), or implanted "tattoos" that are interrogated transdermally (Bandodkar et al. 2015).
The majority of fluorescent chemosensors and biosensors do not undergo changes in emission spectral shape upon analyte binding and accordingly evince monochromatic intensity changes, rather than the dichromatic responses required for ratiometric sensing (de Lorimier et al. 2002). The present subject matter provides methods for converting monochromatic responses into dichromatic responses that enable ratiometric sensing. In embodiments, these methods are based on establishing non-geometrically modulated Forster Resonance Energy Transfer (ngmFRET) between the monochromatic fluorophore (directly responsive partner), and a second fluorophore that neither interacts directly with the ligand, nor is sensitive to ligand-mediated changes in its environment (indirectly responsive partner).
Unlike tgmFRET-based chemical sensing systems (Valeur 2012), this arrangement does not rely on analyte-mediated geometrical changes (inter-fluorophore distance or angle) between the donor and acceptor, but instead exploits effects by analyte binding, which alter the photophysics of only the directly responsive partner such as changes in its spectral properties and non-radiative decay rates (FIG. 3).
The exemplary and non-limiting studies described herein demonstrate how these ngmFRET effects were used to convert monochromatic into dichromatic responses and thereby improve the ratiometric properties of dichromatic responses, using three classes of examples that illustrate the application of this technique both to biosensors and chemosensors (FIG. 4):

1. The analyte recognition element is a protein that undergoes an analyte-mediated conformational change that is alters the properties of an environmentally responsive directly responsive partner (FIG. 4A): a glucose-binding protein in which a conjugated directly responsive fluorophore respond via a glucose-induced protein conformational change that alters its emission properties. This fluorophore is paired with a second, indirectly responsive partner attached to a fusion domain (such as a fluorophore attachment motif attached to, e.g., the first or the last amino acid of the ligand-binding protein). The resulting ngmFRET
established between the two partners converts a monochromatic response of the directly responsive partner into a dichromatic response, or improves its dichromatic, ratiometric properties.
2. The analyte recognition element is a rigid protein with an analyte-binding site located adjacent to an fluorophore (having fluorescence by itself and regardless of the presence of any other fluorophore/partner; FIG. 4B): the monochromatic response of the fluorophore to chloride ion binding in a yellow fluorescent protein is converted to a dichromatic response using a indirectly responsive extrinsic fluorophore site-specifically attached to the protein surface.
3. The analyte recognition element is a synthetic chemoresponsive fluorophore (FIG.
4C): an adaptor protein is engineered to establish ngmFRET between two, site-specifically attached extrinsic fluorophores. The monochromatic response of the directly responsive partner to proton binding is converted into a dichromatic signal.
The first example represents a large class of protein-based fluorescent biosensors which undergo ligand-mediated conformational changes that alter the local environment of an attached fluorophore. Such conformational changes are found in many proteins;
coupling these to fluorescent responses therefore provides a rich source for engineering fluorescent biosensors. For instance, the glucose-binding protein used in this example is a member of the bacterial periplasmic-binding protein (PBP) superfamily which combines a large diversity of ligand specifities with a common structural mechanism (Bemtsson et al. 2010) that is well suited to the construction of fluorescent sensors (de Lorimier et al. 2002, Grunewald 2014).
However, in these proteins, engineered fluorescent responses are more commonly monochromatic than dichromatic. This first example (FIG. 4A) therefore demonstrates how ngmFRET can be used to improve the success rate for engineering ratiometric biosensors that exploit ligand-mediated protein motions. Non-limiting examples of ligand-binding proteins include proteins that bind sugars (such galactose-binding proteins, lactose-binding proteins, arabinose-binding proteins, ribose-binding proteins, and maltose-binding proteins), urea-binding proteins, bicarbonate-binding proteins, phosphate-binding proteins, sulfate-binding proteins, calcium-binding proteins, dipeptide-binding proteins, amino acid-binding proteins (such as histidine-binding proteins, glutamine-binding proteins, glutamate-binding proteins, and aspartate-binding proteins), and iron-binding proteins.
The second example (FIG. 4B) represents a smaller set of proteins that contain fluorophores formed by a self-catalyzed cyclization of a peptide within their sequences such as Green Fluorescent Protein, its engineered variants and homologs(Tsien 1998, Zimmer 2002). Some of these fluorophores function as direct natural chemosensors by interacting with ligands such as protons and halides (Miesenbock, De Angelis and Rothman 1998, Grimley et al. 2013), but typically evince only monochromatic responses. The example demonstrates how ratiometic sensing mechanisms can be engineered into these proteins.
The third example (FIG. 4C) illustrates how protein engineering was used to improve the properties of synthetic directly responsive chemosensors, by incorporating them as extrinsic fluorophores into a protein and combining with a second fluorophore to introduce ngmFRET. In this example, the protein therefore functions as an "adaptor"
scaffold/compound that enables facile integration of multiple functionalities.
Although many small-molecule fluorescent chemosensors have been developed that measure a wide variety of ligands, prior to the invention it was challenging to engineer dichromatic responses. The use of an adaptor protein provides a facile route to integrate multiple fluorophores to generate a dichromatic sensor construct, enabling the power of monochromatic chemosensors to be harnessed and optimized.
In the third example, a chemoresponsive (directly responsive) fluorophore may be linked to another fluorophore (an indirectly responsive fluorophore) using virtually any polypeptide sequence. Though a pH-sensitive chemoresponsive fluorophore is exemplified in the examples, any other chemoresponsive fluorophore may be used. The principles demonstrated for converting the monochromatic response of fluorescein to protons into a dichromatic signal by incorporation into a dually labeled adaptor protein can be extended to other chemoresponsive fluorophores for the detection of a wide variety of analytes. A well-known thiol- or amine-reactive functional group (imidoesters, NHS esters, carbodiimides, maleides, aziridines, arcyloyls; see, e.g., G.T. Hermanson, 2013, Bionjugate Techniques, Academic Press, incorporated herein by reference) may be incorporated into the chemoresponsive fluorophore such that the chemoresponsive fluorophore can be coupled to the adaptor protein (M.S.T. Goncalves, 2009, Chem. Rev., 190-212).
Chemoresponsive sensors can bind specifically to small molecules such as ions, monosaccharides, amino acids, and short peptides, using a variety of molecular recognition units. Such units are then linked to a fluorescent group, the properties of which are altered upon binding the ligand (A. P.
Demchenko, 2015, Introduction to Fluorescence Sensing, Springer). A variety of schemes can be used to couple proton-binding groups such as amines to fluorescence responses (op.
cit.). Fluoresccent chemosensors have been developed for toxic metals such as lead, cadmium, and mercury (K.P. Carter et al., 2014, Chem. Rev., 114, 4564-4601).
Chemoresponsive fluorophores have been developed for glucose (X.S. Sun, T.D.
James, 2015, Chem. Rev., 115, 8001-8037), and other organic analytes including amines, urea, and guanidinium (T.W. Bell and N.M. Hext, 2004, Chem. Soc. Rev., 33, 589-598).
The fluorescent glucose sensor described in the first example has utility in glucose monitoring is essential for the management of diabetes mellitus, a disease that affects at least 366 million people world-wide(Yoo and Lee 2010, Cash and Clark 2010) and is increasing every year. The majority of current glucose-monitoring technologies rely on enzymes for which glucose is one of the substrates(Wang 2008, Bergel, Souppe and Comtat 1989).
Glucose concentration measurements therefore are subject to variations in second substrate concentrations consumed in the enzyme reaction, such as oxygen in the case of glucose oxidase(Tang et al. 2001). Additional complications arise in systems where reaction rates are measured for enzymes immobilized on electrodes. In such arrangements, accuracy is compromised by factors that alter the rate at which glucose arrives at the electrode surface interfere with accuracy, such as hematocrit levels(Karon et al. 2008, Tang et al. 2000), or surface "fouling" by deposition of proteins and cells in the foreign body response(Koschwanez and Reichert 2007, Gifford et al. 2006, Wisniewski and Reichert 2000). Ratiometic fluorescent glucose sensors obviate these problems, and accordingly have been incorporated successfully in optodes for continuous glucose monitoring in animals and humans.
Determination of the concentration of chloride, an essential electrolyte, is a routine measurement in clinical chemistry carried out using potentiometric or optical methods(Huber 2001), and has applications to environmental chemistry(Huber 2000) and corrosion control(de Graaf 2015) as well. Development of a fluorescent, ratiometric chloride biosensor, such as described in the second example enables chloride sensing to be incorporated into point-of-care devices and continuous monitoring systems.
The determination of proton concentrations is necessary for a broad range of applications, ranging from clinical chemistry to environmental science.
Accordingly, the development of fluorescent and other optical probes is an active area(Han 2010, Wencel 2014).
Together these examples demonstrate that the ngmFRET mechanism is applicable to the engineering of a wide variety of semi-synthetic protein-based fluorescent biosensors by combining properties unique to proteins with those of synthetic fluorophores.
Akin to natural cofactors, the fluorophores endow the proteins with functions that cannot be encoded in amino acids, and the proteins modulate the properties of the fluorophores. As illustrated by the analytes chosen in the examples, such engineered fluorescent biosensors have many potential applications, including medical diagnostics.
Mechanisms for Ligand Sensing using Non-Geometric Modulation of FRET
The subject matter disclosed herein is not limited to or bound by any particular scientific theory. However, the discussion below is provided to facilitate the understanding of possible mechanisms involved with ngmFRET signaling in various embodiments described herein. Equations for calculating various values mentioned herein are also provided.
The total signal, S, of a fluorescent sensor (either single-wavelength emission intensities, IA, or ratios of intensities at two wavelengths, R12) is the sum of the fluorescence due to the ligand-free (apo) and ligand-bound states:
S = 4- )+ g 1 where a and ig are the fluorescent baselines in the ligand-free and -bound states, respectively, and is the fractional occupancy of the binding sites. For a single binding site is given by [L]
y 2 - = r 1 Lid + K d where [L] is the ligand (analyte) concentration and Kd the dissociation constant corresponding to an apparent, aPPKd, or true, "e1Cd, value for fits to R12 and IA
respectively.

Fluorescence quantum yields are the fractions of photons emitted by the excited state relative to the total absorbed, and correspond to the ratio of the radiative decay rate relative to the sum of the rates of all possible decay pathways (FIGS. 3A-D). For a single fluorophore:
kr Q= kr ___ + kn, 3 where kr and km are the radiative and non-radiative decay rates of the excited state, respectively. If we define q as the ratio between the radiative and non-radiative decay rates, km q =. ¨ 4 Icr then the quantum yield can be written as = 5 q+1 Chemical sensors exploit the ligand-mediated shift of a fluorescent system between the ligand-free and ligand-bound states which each exhibit distinct quantum yields:
Qobs = Qapo(1¨ .0+ Qsatii 6 where Qobs, Qapo and Qsat are the quantum yield of the total system, the apo-protein, and the ligand-bound complex, respectively. In a system involving energy transfer between a donor and acceptor fluorophore, the Qapo and Qsat quantum yields each are combinations of their respective donor and acceptor quantum yields:
Qapo=D Qapo+A Qapo and 0 ---sat=D Qsat+AQsat 7 where the superscripts D and A indicate donor and acceptor fluorophores respectively. To understand ngmFRET-based sensors, we therefore need to examine the factors that affect each of these four quantum yields.
The intensity of the light emitted by a donor or its acceptor is determined by the rate of photon emission from their respective excited states (FIG. 3A). The excited state of a donor is formed by the incident light from the excitation source, and there are three pathways by which this state decays: radiative and non-radiative decay and resonance transfer (by itself and regardless of the presence of any other fluorophore/parter). By contrast, the rate of formation of the acceptor excited state is determined by the resonance transfer rate from the donor, and there are only two processes that determine its decay rate: the radiative and non-radiative pathways (by itself and regardless of the presence of any other fluorophore/parter).
In an ngmFRET system, the patterns of ligand-mediated fluorescence intensity changes therefore depend on whether the fluorophore that responds directly to ligand binding functions as a donor or acceptor. To understand these relationships, we analyzed the factors that determine the rates of formation and decay of the donor and acceptor excited states.
The rate of resonance energy transfer, kt, along a non-radiative pathway between donor and acceptor (FIG. 3A) is a fraction of the donor radiative emission pathway rate (by itself and regardless of the presence of any other fluorophore/parter), D kr (the emission rate in the absence of an acceptor) multiplied by the energy transfer coupling factor, 0, (Lakowicz 2006, Valeur 2012):
kf=coQDDkr 8 where QD is the donor quantum yield in the absence of an acceptor.
According to the Forster model of weakly coupled oscillators (Lakowicz 2006, Valeur 2012), the energy transfer coupling factor is dependent on the spectral overlap, J, of the donor emission, Dile,õ and acceptor excitation spectrum, A2,,,, and the variation of the geometry, G, between the donor and acceptor excited state transition dipoles with distance, r, and orientation factor, K:
\ 90001n10 co = GO - ,K)J(D /Ie., A
/ler ) 9 1287r5N An 4 where GO - ,K)= 10 r and J(D2em,A2)=.1.F(D2eni(A2),14d2 11 with n the refractive index of medium, NA Avogrado's number, fi(Dkem) the normalized donor emission spectrum, and c(A2,x) the absorption coefficient of the acceptor excitation spectrum [this analysis is a re-arrangement of the traditional presentation of the equations describing tgmFRET, separating the different contributions (geometry, spectral overlap, quenching)]. Ligand-mediated modulation of r, K and J therefore affects kt (Fig. 3b-d), leading to changes in donor and acceptor emission intensities (see below).
At steady state, the concentration of the donor excited state, [D*], is given by the following rate balance equation (see Fig. 3a):
Nook ¨ [D* IDIcnr+Dkr + Icf) = 0 12 where No is the population of ground state fluorophores, kex the rate of excitation photon absorption, a the effective illumination, kt, the resonance energy transfer rate, Dknr and Dkr the radiative and non-radiative decay rates of the donor (by itself and regardless of the presence of any other fluorophore/parter) in the absence of acceptor, respectively.
Substituting Dkr(d +1) for Dkr+Dknr (using equation 4, with dq, the ratio of non-radiative to radiative decay rates in the donor), and replacing kt with equation 8 (with QD =0+ d), according to equation 5), we obtain Nook, ¨[Dtkr (1+ d + ___ 9 1+ dj= 0 13 Hence [D]= N 0akex 14 Dkr(l+d+ 9 j 1+d The intensity of the emitted donor light, ID, is I D 4114 N7. = 0ak ex 15 1+d The donor quantum yield, QD, is this emission intensity relative to the intensity of the excitation, kexallo QD =/ 16 (1+ d + 9 ) 1+d The rate balance equation for the acceptor excited state concentration, [A*], is given by [D*], _ [A * jAkr Akrir ) 17 Consequently, by applying equations 5, 8 and 16, the acceptor quantum yield, QA, is QA= 9 18 (1+41+41+d+ 9 j 1+d where a is the ratio of the radiative and non-radiative pathways in the acceptor.
The ratio of the acceptor and donor quantum yields therefore is QA = 9 19 QB (1+ d)(1 a) This equation clearly shows that a ligand-mediated change in energy transfer (0) or a change in the ratio of radiative to non-radiative emission rates of either the donor (d) or acceptor (a) leads to a change in the ratio of donor and acceptor emission intensities, thereby enabling ratiometry.
Classical ligand-mediated modulation of tgmFRET (traditional geometric FRET -) is concerned only with ligand-mediated changes in the distance between the donor and acceptor (Clegg 1995, Cheung 1991), and does not take advantage of effects that alter the photophysics of individual fluorophores. By contrast, in ngmFRET systems, the directly responsive partner (DRP) responds to ligand binding through ligand mediated changes that alter the ratio of its radiative and non-radiative pathways (quenching, d or a) or its spectral properties (J), whereas the indirectly responsive partner (LRP) changes only as a consequence of the effect that such change have on the resonance energy transfer rate (kt). It is important to realize that the DRP can function either as a ngmFRET donor an acceptor, depending on how the spectral overlap is set up with the IRP. Regardless of whether the DRP
is a donor or acceptor, ligand-mediated alteration of its non-radiative to radiative decay rate ratio (parameter d for a DRP donor; a for an acceptor; by itself and regardless of the presence of any other fluorophore/parter) changes its emission intensity. In DRP donors quenching also alters the ngmFRET transfer rate (see equations 8 and 13), thereby changing the emission intensities of not only itself but also its IRP. By contrast, in DPR acceptors quenching does not alter ngmFRET, and hence do not affect its lRP donor intensity. A DRP
acceptor therefore can alter intensities of its donor IRP only if ligand binding changes 0. If the DRP is a donor, then manipulation of the ngmFRET coupling factor, 0, changes the rate of excited state decay; if it is an acceptor, the rate of excited state formation is altered.
Regardless of whether the DRP is a donor or acceptor, a change in any of the two parameters (0 and d or a) alters the ratio of the donor and acceptor quantum yields (equation 19), thereby enabling ratiometry. Ligand-mediated donor DRP quenching affects the quantum yields of both the donor, QD, and acceptor, QA, quantum yields (equations 16, 18).
Quenching of an acceptor DRP alters only QA (equation 16). Changes in 0 affect quantum yields of both fluorophores, regardless whether the DRP functions as the donor or acceptor (equations 9-11, 16, 18). For systems in which there is no ligand-mediated change in the (average) distance between the two fluorophores, 0 changes only if the DRP
switches between two different excited state populations ("dipole switching") in response to ligand binding and if the two excited states differ in their spectral properties (emission for donor DRPs; absorption for acceptor DRPs). Excited state dipoles usually also differ in their dipole orientations, so it is likely that changes in spectral overlap involve (re-)orientation effects.
They are also likely to differ in the relative rates of their radiative and non-radiative decay rates. Dipole switching therefore is likely to involve a combination of changes in ngmFRET
and quenching effects.
There are eight possible combinations of ligand-mediated changes in quenching and ngmFRET parameters, which have different outcomes on the two emission intensities and their ratio, depending on whether the DRP is the donor or acceptor. The qualitative behavior of the resulting sixteen possibilities in ngmFRET systems are shown in Table 1. Twelve of these have a predictable outcome on the direction of change in the ratio of the two emission intensities. The effect on the direction of change for both donor and acceptor emission intensities can be predicted for seven models. For the other models, the direction of change of one or both peaks depends on the size of the change in the underlying parameters. Purely geometric effects (changes in inter-dipole distance or orientation) always result in anti-correlated changes in emission intensity changes (i.e. one increases and the other decreases, or vice versa). Correlated (i.e. both intensities increase or decrease) or uncorrelated (one changes, the other remains constant) intensity changes therefore are prima facie evidence for an ngmFRET effect.

Table 1. Qualitative analysis of the patterns of donor and acceptor emission intensity changes in ngmFRETa Directly responsive partner Model QA/QD QD QA
Donor 4+ 1' .1, 1' d 0- .1, 1' .1, ctO
d+0+ .1, *
6' 0" sl, * 4, d 0 T T T
do+ T * ,i, d 0" "r *
Acceptor a 0+ 1' si, *
a 0" 4, 1' *
a+0 si, 0 si, a+ 0+ *
a+ si, 1' *
6/0 1' 0 1' a", 1' .1, 1' a" 0" 1' *
aThe effects of increasing or decreasing quenching in the directly responsive ngmFRET
partner (d for donors, a for acceptors) or the energy transfer coupling (0) between the donor and acceptor are tabulated. The consequences of using a directly responsive donor or acceptor are examined. Changes in quenching and energy transfer coupling parameters can occur singly or in combination, leading to 16 possible models. The models examine the effects of the direction of change in quenching parameters (no change, d or a0; increase di- or a+; decrease, d or d) and the energy transfer coupling factor (no change, 05 ;
increase, 0+;
decrease, 0") on the patterns in the direction of change of the donor, QD
(equation 16) or acceptor, QA (equation 18) quantum yields, and their ratio, QA/QD (equation 19): 1', increase;
, decrease; 0, no change; *, response is dependent on precise quantitation rather than direction of change in the underlying parameter values.
Exemplary Dual Labeling Techniques to Construct Donor-Acceptor Pairs The directly responsive fluorophore needs to be site-specifically attached to a site in the protein where it can respond to the ligand-binding event (the one exception is the case where the directly responsive fluorophore is integral to the protein itself, as is the case for YFP). Site-specific attachment of the second, passive fluorophore also is desirable, because it establishes a unique ngmFRET interaction, whereas random attachment gives rise to multiple interactions, only some of which are likely to be result in usable signals with the others contributing to background.

Cysteine thiols provide a convenient, chemically unique functionality for site-specific attachments at positions defined by the protein sequence. Furthermore, straightforward, reversible protection strategies have been developed that enable multiple thiols to be labeled orthogonally at independently addressable sites in successive reactions (Smith et al. 2005).
In this scheme, two labeling sites can be engineered using fusions that combine two proteins or domains containing a single thiol and disulfide bridge, respectively (FIG.
5). In such constructs, labeling proceeds in two steps. First, the unprotected thiol is modified with the first fluorophore under conditions in which the disulfide bridge is fully formed protecting the second thiols. After this labeling step is complete, unincorporated first fluorophore is removed from the reaction. In the second labeling step the thiols in the disulfide bridge are deprotected by reduction, enabling attachment of the ngmFRET partner fluorophore at their positions.
Typically, the single cysteine is located in a protein that binds the analyte of interest (e.g. the periplasmic glucose-binding protein) for coupling of the directly responsive fluorophore, and the disulfide is located in a small domain or protein fused at the N- or C-terminus for attachment of the indirectly responsive ngmFRET partner. In the examples shown here we have used an 17-residue peptide, PZif, derived from a zinc finger protein(Smith et al. 2005) as the disulfide-containing domain. The disulfide-containing protein thioredoxin from Escherichia co/i(Holmgren 1985, Qi 2005, Katti 1990), ecTRX, was also used as a fusion partner. The ecTRX also has been used to construct the adaptor protein by installing a single cysteine mutation that introduces an unprotected thiol on the surface of the protein (see below).
Thiols are not the only means to establish orthogonal chemistries. For example, amines also have a highly selective chemistry. To demonstrate this approach, an unusual version of ecTRX was created in which the amino acid alphabet was limited to 19 residues, eliminating all lysines. This leaves only one reactive primary amine in the protein: the amino terminus. In this manner, constructed a doubly labeled protein by adding the first label to the amino terminus, and the second to the reduced disulfide bridge. A label may be conjugated to ecTRX using a variety of linkers or bonds, including (but not limited to) a disulfide bond, an ester bond, a thioester bond, an amide bond, or a bond that has been formed by a click reaction.

Biosensors Biosensors are molecular recognition elements that transduce ligand-binding events into physical detectable signals. Biosensors as detailed herein bind at least one ligand and emit a signal. A ligand-bound biosensor results in a signal that is different from the unbound biosensor. This difference facilitates detection of the at least one ligand and/or determination of ligand concentration. The biosensors may be used alone, i.e., without the presence or assistance of other reagents.
Described herein are reagentless biosensors engineered to produce a detectable ratiometric detection signal. These biosensors may have altered ligand-binding affinities, tailored ligand-binding specificities, and/or temperature dependencies of ligand binding or stability compared to corresponding naturally occurring ligand-binding proteins. For example, the herein described engineered ligand biosensors provide high-accuracy information related to extended ligand concentration ranges.
Binding of ligand mediates conformational changes in the biosensor, such as hinge-bending motions of the polypeptide. The conformational changes affect the environment of the reporter such that a change in the reporter-generated signal occurs. That is, without ligand bound, the biosensor results in signal generated from the reporter, and when ligand is bound, the signal generated from the reporter changes. The ligand-bound biosensor results in a reporter-generated signal that is different from the unbound biosensor.
Among the advantages of these fluorophone-containing protein constructs is their high durability. The constructs retain their ability to bind ligand, change shape and thus detect the ligand (a) even when immobilized (directly or indirectly) onto a solid surface such as a bead, plate, or sheet; (b) even after desiccation (and subsequent reconstitution in a physiological buffer solution); (c) even when subjected to ambient conditions, e.g., conditions that can be encountered in storage and/or transportation; and (d) even when aged/stored for extended periods of time, e.g., weeks, months, or even years.
Thus, the biosensors do not require refrigeration or a cold chain for distribution, permitting a wider range of applicability such as in-the-field use and reducing the cost of the sensor product.
For clinical applications, microliter volumes, e.g., 10,9, 8, 7, 6, 5, 4, 3, 2, 1, 1.1.1 or less of a bodily fluid may be used. In some embodiments, the volume of sample that is applied to a biosensor or a device comprising a biosensor is less than 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 25, 50, 75, 100, 150, 300, 500, or 1000 pl. In some embodiments, the volume is about 0.1 1.11 to about 1000 Ill, about 0.1 1.11 to about 100 Ill, about 1 1.11 to about 1000 Ill, about 1 p,1 to about 10 Ill, about 1 1.11 to about 100 0, about 1 1.11 to about 50 ta, about 10 tato about 50 0, or about 5 ta to about 50 0. Moreover compared to conventional enzyme-based or antibody based assay systems, the results are achieved virtually instantaneously, e.g., within 30-60 seconds. A further advantage is that the sensors consistently and reliably bind to and detect the analyte in complex fluids such as whole blood. Thus in a clinical setting, whole blood need not be processed, thereby reducing time and cost of the diagnostic procedure.
In non-clinical situations, e.g., industrial of commercial settings such as analysis of waste water, food or beverage production, or bioreactor/fermentation monitoring, the samples to be analyzed can be used directly upon sampling without further purification or processing, similarly reducing time and expense of the test. Moreover, the immobilized sensors need not be washed to remove unbound material following contacting the test sample with the sensors, because the unbound material ("contaminants") do not materially affect the production of a precise, reliable detectable assay signal.
In some embodiments, the methods and compositions include a plurality of a single type of biosensor. The biosensors may be identical in structure and function. For example, the biosensors of a single type may have the same polypeptide, the same reporter, and the same ligand affinity.
In other embodiments, the methods and compositions include a plurality of different types of biosensors. A plurality of these different types of biosensors may be arranged or incorporated in a panel. As used herein, a "panel" refers to two or more biosensors. The two or more biosensors may be different from each other. The biosensors may differ in structure and/or function. Biosensors may differ in polypeptide sequence, reporter, ligand affinities, or a combination thereof. Accordingly, there may be different types of biosensors. In some embodiments, each biosensor in the panel comprises the same reporter group. In some embodiments, each biosensor in the panel comprises a different reporter group. The panel may include at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, or at least 100 biosensors.
The panel of biosensors includes at least one sensor element. "Sensor element"

refers to a single spot, site, location, or well for the at least one biosensor, to which a sample or aliquot thereof may be applied. The panel may be a composite sensor or an array.
In some embodiments, the panel is a composite sensor. In a composite sensor, each sensor element includes a mixture of two or more different biosensors. In some embodiments, the composite sensor includes one sensor element. In some embodiments, the composite sensor includes two or more sensor elements. In some embodiments, signals are measured from a composite sensor in which the signals arise from one or more biosensors in the sensor element. For example, signals may be measured from a composite sensor in which the signals arise from a subset of the total number of biosensors in the sensor element. For example, signals may be measured from a composite sensor in which the signals arise from two of five biosensors in the sensor element.
In some embodiments, the panel is an array. In an array, each sensor element includes a single type of biosensor. An array comprises a plurality of individually and spatially localized sensor elements. Each sensor element includes a biosensor that is different than or the same as the biosensor of a different sensor element. In some embodiments, signals are measured from an array in which the signals arise separately from two or more selected biosensors in separate sensor elements. An array may comprise a plurality of sensor elements of a variety of sizes and configurations. An array may comprise a plurality of sensor elements arranged linearly. For example, an array may comprise a plurality of micrometer-sized sensor elements arranged in a single row. An array may comprise a plurality of sensor elements arranged in a grid. The grid may be two- or three-dimensional. In some embodiments, the grid is a spatially addressable grid. In some embodiments, the biosensors are incorporated into an array, such as a multichannel or multiplexed array.
The biosensors of the present disclosure can be used in any setting where ligand detection is required or desired, such a medical setting (e.g., determining the level of blood ligand in a subject), environmental setting (e.g., determining the level of ligand in an environmental sample), biological setting (e.g., determining the presence or amount of ligand in a reaction), or in process engineering, such as monitoring the amount of ligand in a fermentation reaction (e.g., beer/wine production, etc.). Other examples include, but are not limited to, uses in the food industry (Suleiman et al, In: Biosensor Design and Application:
Mathewson and Finley Eds; American Chemical Society, Washington, DC 1992, vol.
511); in clinical chemistry (Wilkins et al., Med. Eng. Phys. 1996, 18, 273-288; Pickup, Tr. Biotech.
1993, 11, 285-291; Meyerhoff et al., Endricon 1966, 6, 51-58; Riklin et al., Nature 1995, 376, 672-675); Willner et al., J. Am. Chem. Soc. 1996, 118, 10321-10322); as the basis for the construction of a fluorescent flow cell containing immobilized ligand binding protein-FAST
conjugates (see, e.g., Wilkins et al., Med. Eng. Phys. 1966, 18, 273-288;
Pickup, Tr. Biotech.
1993, 11, 285-291; Meyerhoff et al., Endricon. 1966, 6, 51; Group, New Engl.
J. Med. 1993, 329, 977-986; Gough et al., Diabetes 1995, 44, 1005-1009); and in an implantable devices.
The biosensors as detailed herein may be administered in a variety of ways known by those of skill in the art, as appropriate for each application. Biosensors may be provided in a solution. The solution may be buffered. Biosensors may be provided in a solution and mixed directly with a sample. In some embodiments, a biosensor is immobilized onto a surface.
Biosensors may be immobilized within a disposable cartridge into which a sample may be introduced or applied. Biosensors may be implanted or incorporated in a wearable device.
The biosensor may be provided as an optode.
The biosensor may be attached to or incorporated in a wearable device.
Wearable devices may include, for example, adhesive strips, patches, and contact lenses. The biosensor may be configured for placement in contact with a subject's skin or mucosal surface. In some embodiments, the biosensor is configured as an adhesive strip. In some embodiments, the biosensor is configured within or on the surface of a contact lens. In some embodiments, the contact lens is formed from a transparent substrate shaped to be worn directly over a subject's eye, as described in, for example, U.S. Patent No. 8,608,310.
The biosensor may be implanted. The biosensor may be implanted in a subject's body. The biosensor may be implanted in a subject's blood vessel, vein, eye, natural or artificial pancreas, skin, or anywhere in the alimentary canal including the stomach, intestine and esophagus. The biosensor may be implanted in a subject with a microbead.
In some embodiments, the biosensor is configured to be implanted in the skin. The biosensor may be implanted in a subject sub-dermally. The biosensor may generate the signal trans-dermally.
In some embodiments, the biosensor may be implanted in a subject with transdermal microbeads, wherein the optical signals can be transmitted remotely between the biosensor and detecting device.
In some embodiments, the biosensor is administered as an optode. As used herein, "optode" refers to an optical fiber with a single biosensor, or a composite biosensor, immobilized at the surface or at the end. An "optode" may also be referred to as an "optrode." In some embodiments, the biosensor is implanted in a subject as an optode. The optode may be incorporated with or into a needle. The optode may be incorporated with a probe such as endoscopy or colonoscopy probes. The optode may be used in a tumor, near a tumor, or at the periphery of a tumor. In some embodiments, the biosensor may be implanted in a subject as an optode, wherein the optical signals can be transmitted between the biosensor and detecting device using physical links. In some embodiments, the biosensor is administered as an optode to a sample or reaction. The optode may be contacted with a sample or reaction. In some embodiments, an optode is used to continuously or episodically monitor a ligand in a sample or reaction.
Methods Of Detecting The Presence Of A Ligand Provided herein is a method of detecting the presence of a ligand in a sample.
The method may include contacting the biosensor with the sample; measuring a signal from the biosensor; and comparing the signal to a ligand-free control. A difference in signal indicates the presence of ligand in the sample.
Also provided herein is a method of detecting the presence of ligand in a sample. The method may include (a) providing a ligand biosensor disclosed herein in which the reporter group is attached the ligand so that a signal transduced by the reporter group when the ligand is bound to ligand differs from a signal transduced by the reporter group when the ligand is not bound to ligand; (b) contacting the biosensor with the test sample under conditions such that the biosensor can bind to ligand present in the test sample; and (c) comparing the signal transduced by the reporter group when the biosensor is contacted with the test sample with the signal transduced by the reporter group when the biosensor is contacted with a ligand-free control sample, wherein a difference in the signal transduced by the reporter group when the biosensor is contacted with the test sample, as compared to when the biosensor is contacted with the control sample, indicates that the test sample contains ligand.
Methods Of Determining The Concentration Of A Ligand Provided herein is a method of determining the concentration of a ligand in a sample.
The method may include contacting the biosensor with the sample; measuring a signal from the biosensor; and comparing the signal to a standard hyperbolic ligand binding curve to determine the concentration of ligand in the test sample. The standard hyperbolic ligand binding curve may be prepared by measuring the signal transduced by the biosensor when contacted with control samples containing known concentrations of ligand.

Another aspect of the present disclosure provides a method of determining the concentration of ligand in a test sample comprising, consisting of, or consisting essentially of:
(a) providing a ligand biosensor comprising a ligand biosensor as described herein in which the reporter group is attached the ligand so that a signal transduced by the reporter group when the ligand is bound to ligand differs from a signal transduced by the reporter group when the ligand is not bound to ligand; (b) contacting the biosensor with the test sample under conditions such that the biosensor can bind to ligand present in the test sample; and (c) comparing the signal transduced by the reporter group when the biosensor is contacted with the test sample with a standard hyperbolic ligand binding curve prepared by measuring the signal transduced by the reporter group when the biosensor is contacted with control samples containing known quantities of ligand to determine the concentration of ligand in the test sample.
Methods Of Monitoring The Presence Of A Ligand The present invention is directed to a method of episodically or continuously monitoring the presence of a ligand in a reaction. In certain embodiments, the biosensors may be used in the continuous monitoring of ligand in a reaction. In certain embodiments, the ligand sensors may be used in episodic monitoring of sample aliquots.
The method of episodically or continuously monitoring the presence of a ligand in a reaction may include contacting the biosensor with the reaction; maintaining the reaction under conditions such that the polypeptide is capable of binding ligand present in the reaction; and episodically or continuously monitoring the signal from the biosensor in the reaction.
The method of episodically or continuously monitoring the presence of a ligand in a reaction may include contacting the biosensor with the reaction; maintaining the reaction under conditions such that the polypeptide is capable of binding ligand present in the reaction; episodically or continuously monitoring the signal from the biosensor in the reaction; and comparing the signal to a standard hyperbolic ligand binding curve to determine the concentration of ligand in the test sample. The standard hyperbolic ligand binding curve may be prepared by measuring the signal transduced by the biosensor when contacted with control samples containing known concentrations of ligand.
In some embodiments, the method further includes comparing the signal to a ligand-free control, wherein a difference in signal indicates the presence of ligand in the reaction.

In some embodiments, the method further includes comparing the signal to a standard hyperbolic ligand binding curve to determine the concentration of ligand in the test sample.
The standard hyperbolic ligand binding curve may be prepared by measuring the signal transduced by the biosensor when contacted with control samples containing known concentrations of ligand.
Another aspect of the present disclosure provides a method of continuously monitoring the presence of ligand in a reaction comprising, consisting of, or consisting essentially of: (a) providing a ligand biosensor as described herein in which the reporter group is attached the ligand-binding protein so that a signal transduced by the reporter group when the ligand is bound to ligand-binding protein differs from a signal transduced by the reporter group when the ligand is not bound to the ligand-binding protein; (b) maintaining the biosensor within the reaction and under conditions such that the biosensor can bind to ligand present in the reaction; (c) continuously monitoring the signal transduced by the reporter group when the biosensor is contacted with the ligand present in the reaction;
and optionally (d) comparing the signal transduced by the reporter group when the biosensor is contacted with the ligand present in the reaction with the signal transduced by the reporter group when the biosensor is contacted with a ligand-free control sample, wherein a difference in the signal transduced by the reporter group when the biosensor is contacted with the ligand present in the reaction, as compared to when the biosensor is contacted with the control sample, indicates ligand is present in the reaction.
Yet another aspect of the present disclosure provides a method of continuously monitoring the concentration of ligand in a reaction comprising, consisting of, or consisting essentially of: (a) providing a ligand biosensor comprising a ligand biosensor as described herein in which the reporter group is attached the ligand so that a signal transduced by the reporter group when the ligand is bound to ligand differs from a signal transduced by the reporter group when the ligand is not bound to ligand; (b) maintaining the biosensor within the reaction under conditions such that the biosensor can bind to ligand present in the reaction; and (c) continuously monitoring the signal transduced by the reporter group when the biosensor is contacted with the ligand present in the reaction; and (d) comparing the signal transduced by the reporter group when the biosensor is contacted with the ligand present in the reaction with a standard hyperbolic ligand binding curve prepared by measuring the signal transduced by the reporter group when the biosensor is contacted with control samples containing known quantities of ligand to determine the concentration of ligand in the reaction.
General Definitions Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, and biochemistry).
As used herein, the term "about" in the context of a numerical value or range means 10% of the numerical value or range recited or claimed, unless the context requires a more limited range.
In the descriptions above and in the claims, phrases such as "at least one of' or "one or more of' may occur followed by a conjunctive list of elements or features.
The term "and/or" may also occur in a list of two or more elements or features. Unless otherwise implicitly or explicitly contradicted by the context in which it is used, such a phrase is intended to mean any of the listed elements or features individually or any of the recited elements or features in combination with any of the other recited elements or features. For example, the phrases "at least one of A and B;" "one or more of A and B;" and "A and/or B"
are each intended to mean "A alone, B alone, or A and B together." A similar interpretation is also intended for lists including three or more items. For example, the phrases "at least one of A, B, and C;" "one or more of A, B, and C;" and "A, B, and/or C" are each intended to mean "A alone, B alone, C alone, A and B together, A and C together, B and C
together, or A
and B and C together." In addition, use of the term "based on," above and in the claims is intended to mean, "based at least in part on," such that an unrecited feature or element is also permissible It is understood that where a parameter range is provided, all integers within that range, and tenths thereof, are also provided by the invention. For example, "0.2-5 mg" is a disclosure of 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg etc. up to and including 5.0 mg.
A small molecule is a compound that is less than 2000 daltons in mass. The molecular mass of the small molecule is preferably less than 1000 daltons, more preferably less than 600 daltons, e.g., the compound is less than 500 daltons, 400 daltons, 300 daltons, 200 daltons, or 100 daltons.

As used herein, an "isolated" or "purified" nucleic acid molecule, polynucleotide, polypeptide, or protein, is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized. Purified compounds are at least 60% by weight (dry weight) the compound of interest. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight the compound of interest. For example, a purified compound is one that is at least 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99%, or 100% (w/w) of the desired compound by weight. Purity is measured by any appropriate standard method, for example, by column chromatography, thin layer chromatography, or high-performance liquid chromatography (HPLC) analysis. A purified or isolated polynucleotide (ribonucleic acid (RNA) or deoxyribonucleic acid (DNA)) is free of the genes/nucleic acids or sequences/amino acids that flank it in its naturally-occurring state.
Purified also defines a degree of sterility that is safe for administration to a human subject, e.g., lacking infectious or toxic agents.
Similarly, by "substantially pure" is meant a nucleotide or polypeptide that has been separated from the components that naturally accompany it. Typically, the nucleotides and polypeptides are substantially pure when they are at least 60%, 70%, 80%, 90%, 95%, or even 99%, by weight, free from the proteins and naturally-occurring organic molecules with they are naturally associated.
The transitional term "comprising," which is synonymous with "including,"
"containing," or "characterized by," is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. By contrast, the transitional phrase "consisting of' excludes any element, step, or ingredient not specified in the claim. The transitional phrase "consisting essentially of' limits the scope of a claim to the specified materials or steps "and those that do not materially affect the basic and novel characteristic(s)" of the claimed invention.
"Subject" as used herein refers to any organism from which a biological sample is obtained. For example, the sample is a biological fluid or tissue. For example, a subject is one who wants or is in need of detecting ligand or determining the concentration of ligand with the herein described biosensors. The subject may be a human or a non-human animal.
The subject may be a mammal. The mammal may be a primate or a non-primate. The mammal can be a primate such as a human; a non-primate such as, for example, dog, cat, horse, cow, pig, mouse, rat, camel, llama, goat, rabbit, sheep, hamster, and guinea pig; or non-human primate such as, for example, monkey, chimpanzee, gorilla, orangutan, and gibbon. The subject may be of any age or stage of development, such as, for example, an adult, an adolescent, or an infant.
As used herein, an "expression vector" is a DNA or RNA vector that is capable of effecting expression of one or more polynucleotides. Preferably, the expression vector is also capable of replicating within the host cell. Expression vectors can be either prokaryotic or eukaryotic, and are typically include plasmids. Expression vectors of the present invention include any vectors that function (i.e., direct gene expression) in host cells of the present invention, including in one of the prokaryotic or eukaryotic cells described herein, e.g., gram-positive, gram-negative, pathogenic, non-pathogenic, commensal, cocci, bacillus, or spiral-shaped bacterial cells; archaeal cells; or protozoan, algal, fungi, yeast, plant, animal, vertebrate, invertebrate, arthropod, mammalian, rodent, primate, or human cells. Expression vectors of the present invention contain regulatory sequences such as transcription control sequences, translation control sequences, origins of replication, and other regulatory sequences that are compatible with the host cell and that control the expression of a polynucleotide. In particular, expression vectors of the present invention include transcription control sequences. Transcription control sequences are sequences which control the initiation, elongation, and termination of transcription. Particularly important transcription control sequences are those which control transcription initiation such as promoter, enhancer, operator and repressor sequences. Suitable transcription control sequences include any transcription control sequence that can function in at least one of the cells of the present invention. A variety of such transcription control sequences are known to those skilled in the art.
As used herein, the singular forms "a," "an," and "the" include the plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a disease,"
"a disease state", or "a nucleic acid" is a reference to one or more such embodiments, and includes equivalents thereof known to those skilled in the art and so forth.
As used herein, "pharmaceutically acceptable" carrier or excipient refers to a carrier or excipient that is suitable for use with humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit/risk ratio. It can be, e.g., a pharmaceutically acceptable solvent, suspending agent or vehicle, for delivering the instant compounds to the subject.

The term "diagnosis" refers to a determination that a disease is present in the subject.
Similarly, the term "prognosis" refers to a relative probability that a certain future outcome may occur in the subject. For example, in the context of the present disclosure, prognosis can refer to the likelihood that an individual will develop a disease, or the likely severity of the disease (e.g., severity of symptoms, rate of functional decline, survival, etc.).
Unless required otherwise by context, the terms "polypeptide" and "protein"
are used interchangeably.
A polypeptide or class of polypeptides may be defined by the extent of identity (%
identity) of its amino acid sequence to a reference amino acid sequence, or by having a greater % identity to one reference amino acid sequence than to another. A
variant of any of genes or gene products disclosed herein may have, e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid or amino acid sequences described herein. The term "% identity," in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection. For example, % identity is relative to the entire length of the coding regions of the sequences being compared, or the length of a particular fragment or functional domain thereof. Variants as disclosed herein also include homologs, orthologs, or paralogs of the genes or gene products described herein. In some embodiments, variants may demonstrate a percentage of homology or identity, for example, at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity conserved domains important for biological function, e.g., in a functional domain, e.g. a ligand-binding or catalytic domain.
For sequence comparison, one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. Percent identity is determined using BLAST. For the BLAST searches, the following parameters were employed: (1) Expect threshold is 10; (2) Gap cost is Existence:11 and Extension:1; (3) The Matrix employed is BLOSUM62; (4) The filter for low complexity regions is "on."

The present invention also provides for functional fragments of the genes or gene products described herein. A fragment of a protein is characterized by a length (number of amino acids) that is less than the length of the full length mature form of the protein. A
fragment, in the case of these sequences and all others provided herein, may be a part of the whole that is less than the whole. Moreover, a fragment ranges in size from a single nucleotide or amino acid within a polynucleotide or polypeptide sequence to one fewer nucleotide or amino acid than the entire polynucleotide or polypeptide sequence. Finally, a fragment is defined as any portion of a complete polynucleotide or polypeptide sequence that is intermediate between the extremes defined above.
For example, fragments of any of the proteins or enzymes disclosed herein or encoded by any of the genes disclosed herein can be 10 to 20 amino acids, 10 to 30 amino acids, 10 to 40 amino acids, 10 to 50 amino acids, 10 to 60 amino acids, 10 to 70 amino acids, 10 to 80 amino acids, 10 to 90 amino acids, 10 to 100 amino acids, 50 to 100 amino acids, 75 to 125 amino acids, 100 to 150 amino acids, 150 to 200 amino acids, 200 to 250 amino acids, 250 to 300 amino acids, or 300 to 350 amino acids. The fragments encompassed in the present subject matter comprise fragments that retain functional fragments. As such, the fragments preferably retain the binding domains that are required or are important for functional activity. Fragments can be determined or generated by using the sequence information herein, and the fragments can be tested for functional activity using standard methods known in the art. For example, the encoded protein can be expressed by any recombinant technology known in the art and the binding activity of the protein can be determined.
As used herein a "biologically active" fragment is a portion of a polypeptide which maintains an activity of a full-length reference polypeptide. Biologically active fragments as used herein exclude the full-length polypeptide. Biologically active fragments can be any size as long as they maintain the defined activity. Preferably, the biologically active fragment maintains at least 10%, at least 50%, at least 75% or at least 90%, of the activity of the full length protein.
Amino acid sequence variants/mutants of the polypeptides of the defined herein can be prepared by introducing appropriate nucleotide changes into a nucleic acid defined herein, or by in vitro synthesis of the desired polypeptide. Such variants/mutants include, for example, deletions, insertions or substitutions of residues within the amino acid sequence. A
combination of deletion, insertion and substitution can be made to arrive at the final construct, provided that the final peptide product possesses the desired activity and/or specificity.
Mutant (altered) peptides can be prepared using any technique known in the art. For example, a polynucleotide defined herein can be subjected to in vitro mutagenesis or DNA
shuffling techniques as broadly described by Harayama (1998). Products derived from mutated/altered DNA can readily be screened using techniques described herein to determine if they possess, for example, ligand binding activity.
In designing amino acid sequence mutants, the location of the mutation site and the nature of the mutation will depend on characteristic(s) to be modified. The sites for mutation can be modified individually or in series, e.g., by (1) substituting first with conservative amino acid choices and then with more radical selections depending upon the results achieved, (2) deleting the target residue, or (3) inserting other residues adjacent to the located site.
Amino acid sequence deletions generally range from about 1 to 15 residues, more preferably about 1 to 10 residues and typically about 1 to 5 contiguous residues. In some embodiments, a mutated or modified protein does not comprise any deletions or insertions.
In various embodiments, a mutated or modified protein has less than about 10, 9, 8, 7, 6, 5, 4, 3, or 2 deleted or inserted amino acids.
Substitution mutants have at least one amino acid residue in the polypeptide molecule removed and a different residue inserted in its place. Sites may be substituted in a relatively conservative manner in order to maintain activity and/or specificity. Such conservative substitutions are shown in the table below under the heading of "exemplary substitutions."
In certain embodiments, a mutant/variant polypeptide has only, or not more than, one or two or three or four conservative amino acid changes when compared to a naturally occurring polypeptide. Details of conservative amino acid changes are provided in the table below. As the skilled person would be aware, such minor changes can reasonably be predicted not to alter the activity of the polypeptide when expressed in a recombinant cell.

Exemplary Substitutions Original Residue Exemplary Substitutions Alanine (Ala) Val; Leu; Ile; Gly Arginine (Arg) Lys Asparagine (Asn) Gln; His Cysteine (Cys) Ser Glutamine (Gin) Asn; His Glutamic Acid (Glu) Asp Glycine (Gly) Pro; Ala Histidine (His) Asn; Gln Isoleucine (Ile) Leu; Val; Ala Leucine (Leu) Ile; Val; Met; Ala; Phe Lysine (Lys) Arg Methionine (Met) Leu; Phe Phenylalanine (Phe) Leu; Val; Ala Proline (Pro) Gly Serine (Ser) Thr Threonine (Thr) Ser Tryptophan (Trp) Tyr Tyrosine (Tyr) Trp; Phe Valine (Val) Ile; Leu; Met; Phe; Ala Mutations can be introduced into a nucleic acid sequence such that the encoded amino acid sequence is altered by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. A "conservative amino acid substitution"
is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. Certain amino acids have side chains with more than one classifiable characteristic. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, tryptophan, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tyrosine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in a given polypeptide is replaced with another amino acid residue from the same side chain family.
Alternatively, in another embodiment, mutations can be introduced randomly along all or part of a given coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for given polypeptide biological activity to identify mutants that retain activity.
Conversely, the invention also provides for variants with mutations that enhance or increase the endogenous biological activity. Following mutagenesis of the nucleic acid sequence, the encoded protein can be expressed by any recombinant technology known in the art and the activity/specificity of the protein can be determined. An increase, decrease, or elimination of a given biological activity of the variants disclosed herein can be readily measured by the ordinary person skilled in the art, i.e., by measuring the capability for binding a ligand and/or signal transduction.
In various embodiments, a polypeptide comprises mutations such that 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or less than about 10, 9, 8, 7, 6, 5, 4, 3, or 2 amino acids is substituted with a cysteine and/or a lysine.
Polypeptides can be produced in a variety of ways, including production and recovery of natural polypeptides or recombinant polypeptides according to methods known in the art.
In one embodiment, a recombinant polypeptide is produced by culturing a cell capable of expressing the polypeptide under conditions effective to produce the polypeptide, such as a host cell defined herein.
Key to the Sequence Listing SEQ ID NO Sequence Name 1 C1BP1, also referred to herein as laYFP

ttGGBP.11C.O.bZif (11C substitution mutant with bZif fusion, signal peptide replaced with M and a GGSHHHHHH at C-terminus) 16 ttGGBP.17C.O.bZif (17C substitution mutant with bZif fusion, signal peptide replaced with M and a GGSHHHHHH at C-terminus)

17 ttGGBP.111C.O.bZif (111C substitution mutant with bZif fusion, signal peptide replaced with M and a GGSHHHHHH at C-terminus)

18 ttGGBP.151C.O.bZif (151C substitution mutant with bZif fusion, signal peptide replaced with M and a GGSHHHHHH at C-terminus)

19 ttGGBP.182C.O.bZif (182C substitution mutant with bZif fusion, signal peptide replaced with M and a GGSHHHHHH at C-terminus) ttGGBP.17C.3.Trx (17C, 16N, 211A substitution mutant with ecTRX
fusion, signal peptide replaced with M and a GGSHHHHHH at C-terminus) Trx.ttGGBP.17C.3 (17C, 16N, 211A substitution mutant with ecTRX
21 fusion, signal peptide replaced with M and a GGSHHHHHH at C-terminus) ttGGBP.182C.2.Trx (182C, 69P, 152P substitution mutant with ecTRX
22 fusion, signal peptide replaced with M and a GGSHHHHHH at C-terminus) Trx.ttGGBP.182C.2 (182C, 69P, 152P substitution mutant with ecTRX
23 fusion, signal peptide replaced with M and a GGSHHHHHH at C-terminus) 24 Adaptor Adaptor1.0 26 Adaptor2.0a 27 Adaptor2.0b 28 Adaptor3.0 29 Adaptor4.0 Adaptor5.0 31 Adaptor6.0 32 Adaptor7.0 33 Adaptor8.0 34 Adaptor9.0 Adaptor10.0 36 Adaptor11.0 37 Adaptor12.0 38 Adaptor13.0 39 Adaptor14.0 Adaptor15.0 41 Adaptor16.0 42 PZif 44 Hexahistidine Tag 45 Hexalysine Tag 46 C1BP1 expression construct 47 C1BP2 expression construct 48 C1BP3 expression construct 49 C1BP4 expression construct 50 C1BP5 expression construct 51 C1BP6 expression construct 52 C1BP7 expression construct 53 C1BP8 expression construct 54 C1BP9 expression construct 55 C1BP10 expression construct 56 C1BP11 expression construct 57 C1BP12 expression construct 58 C1BP13 expression construct 59 C1BP14 expression construct 60 ttGGBP.11C.O.bZif expression construct 61 ttGGBP.17C.O.bZif expression construct 62 ttGGBP.111C.O.bZif expression construct 63 ttGGBP.151C.O.bZif expression construct 64 ttGGBP.182C.O.bZif expression construct 65 ttGGBP.17C.3.Trx expression construct 66 Trx.ttGGBP.17C.3 expression construct 67 ttGGBP.182C.2.Trx expression construct 68 Trx.ttGGBP.182C.2 expression construct 69 Adaptor expression construct 70 Adaptor1.0 expression construct 71 Adaptor2.0a expression construct 72 Adaptor2.0b expression construct 73 Adaptor3.0 expression construct 74 Adaptor4.0 expression construct 75 Adaptor5.0 expression construct 76 Adaptor6.0 expression construct 77 Adaptor7.0 expression construct 78 Adaptor8.0 expression construct 79 Adaptor9.0 expression construct 80 Adaptor10.0 expression construct 81 Adaptor11.0 expression construct 82 Adaptor12.0 expression construct 83 Adaptor13.0 expression construct 84 Adaptor14.0 expression construct 85 Adaptor15.0 expression construct 86 Adaptor16.0 expression construct 87 ecGGBP [U.S. National Center for Biotechnology Information (NCBI) Accession No. WP 032329053]
88 ttGGBP (NCBI Accession Nos. YP_003852930.1 and WP_013298803.1) 89 stGGBP (NCBI Accession No. WP_001036943) 90 chyGGBP (NCBI Accession Nos. WP 013402088.1 and YP_003991244.1) 91 cobGGBP (NCBI Accession Nos. WP 013289482.1 and YP_003839461.1) 92 pspGGBP (NCBI Accession Nos. WP 015735911.1 and YP_003243743.1) 93 csaGGBP (NCBI Accession Nos. WP 013273028.1 and YP_003822565.1) 94 bprGGBP (NCBI Accession Nos. WP 013280279.1 and YP_003830205.1) 95 rinGGBP A (NCBI Accession Nos. WP 006855636.1 and YP_007778116.1) 96 fprGGBP (NCBI Accession Nos. WP 015536639.1 and YP 007799070.1) 97 cljGGBP (NCBI Accession No. CLJU_c08950) 98 cauGGBP (NCBI Accession No. CAETHG_2989) 99 rinGGBP B (NCBI Accession Nos. WP 006855628.1 and YP_007778124.1) 100 erhGGBP (NCBI Accession Nos. WP 003775352.1 and YP_004561181.1) 101 ereGGBP (NCBI Accession Nos. WP 012741392.1 and YP_002936409.1) 02 mpUBP; [U.S. National Center for Biotechnology Information (NCBI) Accession Nos. YP 004483096.1 and WP 013797647.1]
03 mhUBP; [U.S. National Center for Biotechnology Information (NCBI) Accession Nos. YP 005430828.1 and WP 014422383.1]
04 bsUBP; [U.S. National Center for Biotechnology Information (NCBI) Accession Nos. YP 006233530.1 and WP 014665698.1]
05 dcUBP; [U.S. National Center for Biotechnology Information (NCBI) Accession Nos. YP 004496535.1 and WP 013809819.1]
06 gtUBP; [U.S. National Center for Biotechnology Information (NCBI) Accession Nos. YP 004588319.1 and WP 013877063.1 ]
07 ctUBP; [U.S. National Center for Biotechnology Information (NCBI) Accession Nos. YP 001038237.1 and WP 003515797.1]
08 csUBP; [U.S. National Center for Biotechnology Information (NCBI) Accession Nos. YP 001181243.1 and WP 011917972.1]
09 taUBP; [U.S. National Center for Biotechnology Information (NCBI) Accession Nos. YP 003473480.1 and WP 012991759.1]
glcUBP; [U.S. National Center for Biotechnology Information (NCBI) Accession Nos. YP 147790.1 and WP 011231423.1]
111 psUBP; [U.S. National Center for Biotechnology Information (NCBI) Accession Nos. YP 003241723.1 and WP 015734090.1]
teUBP; [U.S. National Center for Biotechnology Information (NCBI) Accession No. YP 681910.1 and WP 011567844.1]
13 ttGBP [U.S. National Center for Biotechnology Information (NCBI) Accession Nos. YP 004303.1 and WP 011172778.1]
14 tsGBP [U.S. National Center for Biotechnology Information (NCBI) Accession Nos. YP 004202647.1 and WP 015717367.1]
dmGBP [U.S. National Center for Biotechnology Information (NCBI) Accession Nos. YP 004171760.1 and WP 013557600.1]
6 tnGBP [U.S. National Center for Biotechnology Information (NCBI) Accession Nos. YP 002534202.1 and WP 015919155.1]
17 koGBP [U.S. National Center for Biotechnology Information (NCBI) Accession No. YP 002941687.1 and WP 015869326.1]
8 bhGBP [U.S. National Center for Biotechnology Information (NCBI) Accession Nos. NP 244712.1 and WP 010899970.1]
19 smGBP [U.S. National Center for Biotechnology Information (NCBI) Accession Nos. YP 001041152.1 and WP 011839435.1]

20 asGBP [U.S. National Center for Biotechnology Information (NCBI) Accession No. YP 831349.1 and WP 011691715.1]

21 ttLacBP1 [U.S. National Center for Biotechnology Information (NCBI) Accession No. YP 144032.1]

22 tsLacBP2 [U.S. National Center for Biotechnology Information (NCBI) Accession Nos. YP 004202714.1 and WP 015717434.1 ]
123 toLacBP3 [U.S. National Center for Biotechnology Information (NCBI) Accession Nos. YP 006972155.1 and WP 016329249.1]
24 tsLacBP4 [U.S. National Center for Biotechnology Information (NCBI) Accession Nos. YP 002514099.1 and WP 012638591.1]
25 rdLacBP5 [U.S. National Center for Biotechnology Information (NCBI) Accession Nos. YP 683924.1 and WP 011569849.1]
26 msLacBP6 [U.S. National Center for Biotechnology Information (NCBI) Accession Nos. YP 006556686.1 and WP 014869652.1]
27 tsLacBP7 [U.S. National Center for Biotechnology Information (NCBI) Accession Nos. YP_005654632.1 and WP_014515914.1]
128 maLacBP8 [U.S. National Center for Biotechnology Information (NCBI) Accession Nos. YP 005886720.1 and WP 014578260.1]
29 adLacBP9 [U.S. National Center for Biotechnology Information (NCBI) Accession No. YP_466099.1 and WP_011421944.1]
30 pgLacBP10 [U.S. National Center for Biotechnology Information (NCBI) Accession No. YP_004304976.1 and WP_013653981.1]
31 psLacBP11 [U.S. National Center for Biotechnology Information (NCBI) Accession No. YP_006522676.1 and WP_014851134.1]
32 rsLacBP12 [U.S. National Center for Biotechnology Information (NCBI) Accession Nos. RSP 3372 and YP 354877.1]
33 fsLacBP13 [U.S. National Center for Biotechnology Information (NCBI) Accession Nos. YP_004603455.1 and WP_013886373.1]
34 taLacBP14 [U.S. National Center for Biotechnology Information (NCBI) Accession No. YP_003317968.1]
135 synBicarbBP1 [U.S. National Center for Biotechnology Information (NCBI) Accession Nos. YP_005410477.1 and WP_Ol 0873997.1]
teBicarbBP2 [U.S. National Center for Biotechnology Information (NCBI) Accession No. NP_682790.1]
ctBicarbBP3 [U.S. National Center for Biotechnology Information (NCBI) Accession Nos. YP_007090308.1 and WP_Ol 5152989.1]
calBicarbBP4 [U.S. National Center for Biotechnology Information (NCBI) Accession Nos. YP_007137061.1 and WP_015197735.1]
avBicarbBP5 [U.S. National Center for Biotechnology Information (NCBI) Accession Nos. YP_321546.1 and WP_011317875.1]
140 cmBicarbBP6 [U.S. National Center for Biotechnology Information (NCBI) Accession Nos. YP 007099445.1 and WP 015162006.1]
41 mhFeBP1 [U.S. National Center for Biotechnology Information (NCBI) Accession Nos. YP 007884192.1 and WP 006253500.1]
142 exiFeBP2 [U.S. National Center for Biotechnology Information (NCBI) Accession Nos. YP 002886303.1 and WP 015880417.1]
143 teFeBP3 [U.S. National Center for Biotechnology Information (NCBI) Accession No. NP 681303.1]
144 cnFeBP4 [U.S. National Center for Biotechnology Information (NCBI) Accession Nos. YP 003796723.1 and WP 013247623.1]
145 ttFeBP5 [U.S. National Center for Biotechnology Information (NCBI) Accession No. YP 144894.1]
146 msFeBP6 [U.S. National Center for Biotechnology Information (NCBI) Accession Nos. YP 003686074.1 and WP 013159102.1]
147 srFeBP7 [U.S. National Center for Biotechnology Information (NCBI) Accession Nos. YP 003572493.1 and WP 013062602.1]
148 h1FeBP8 [U.S. National Center for Biotechnology Information (NCBI) Accession Nos. YP 002564837.1 and WP 012659409.1]
149 Yellow Fluorescent Protein 150 Yellow Fluorescent Protein without N-terminal Methionine 151 ecTrx 152 GGSHHHHHH Sequence 153 ecGGBP without signal peptide 154 ttGGBP (with signal peptide replaced with M) 155 stGGBP (with signal peptide replaced with M) 156 chyGGBP (with signal peptide replaced with M) 157 cobGGBP (with signal peptide replaced with M) 158 pspGGBP (with signal peptide replaced with M) 159 csaGGBP (with signal peptide replaced with M) 160 bprGGBP (with signal peptide replaced with M) 161 rinGGBP_A (with signal peptide replaced with M) 162 fprGGBP (with signal peptide replaced with M) 163 cljGGBP (with signal peptide replaced with M) 164 cauGGBP (with signal peptide replaced with M) 165 rinGGBP_B (with signal peptide replaced with M) 166 erhGGBP (with signal peptide replaced with M) 167 ereGGBP (with signal peptide replaced with M) The terms "bZir and "f3Zif" are used synonymously herein.
Exemplary amino acid sequences are listed below for convenience:
ecTrx (SEQ ID NO: 151) MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEY
QGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQL
KEFLDANLA
YFP (SEQ ID NO: 149) MVSKGEELFTGVVP ILVELDGDVNGHKF SVS GEGEGDATYGKLTLKFICT
TGKLPVPWPTLVTTFGYGLQCFARYPDHMKRHDFFKSAMPEGYVQERTIF
FKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHN
VYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNH
YLSYQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYKGGSNDYKDDDD
K
C1BP1 (SEQ ID NO: 1) MVSKGEELFTGVVP ILVELDGDVNGHKF SVS GEGEGDATYGKLTLKFICT
TGKLPVPWPTLVTTFGYGVQCFARYPDHMRQHDFFKSAMPEGYVQERTIF
FKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHN
VYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNH
YLSYQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGSNDYKDDDD
KGGSHHHHHH**
C1BP2 (SEQ ID NO: 2) MVSKGEELFTGVVP ILVELDGDVNGHKF SVS GEGEGDATYGKLTLKFICT
TGKLPVPWPTLVTTFGYGVQCFARYPDHMRQHDFFKSAMPEGYVQERTIF
FKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHN
VYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNH
YLSYQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGSNDYKDDDD
KGGSHHHHHH**
C1BP3 (SEQ ID NO: 3) MVSKGEELFTGVVP ILVELDGDVNGHKF SVS GEGEGDATYGKLTLKFICT
TGKLPVPWPTLVTTFGYGVQCFARYPDHMRQHDFFKSAMPEGYVQERTIF
FKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHN
VYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNH
YLSYQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGSNDYKDDDD
KGGSHHHHHH**
C1BP4 (SEQ ID NO: 4) MVSKGEELFTGVVP ILVELDGDVNGHKF SVS GEGEGDATYGKLTLKFICT
TGKLPVPWPTLVTTFGYGVQCFARYPDHMRQHDFFKSAMPEGYVQERTIF
FKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHN
VYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNH
YLSYQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGSNDYKDDDD
KGGSHHHHHH**
C1BP5 (SEQ ID NO: 5) MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICT
TGKLPVPWPTLVTTFGYGVQCFARYPDHMRQHDFFKSAMPEGYVQERTIF
FKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHN
VYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNH
YLSYQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGSNDYKDDDD
KGGSHHHHHH**
C1BP6 (SEQ ID NO: 6) MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICT
TGKLPVPWPTLVTTFGYGVQCFARYPDHMRQHDFFKSAMPEGYVQERTIF
FKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHN
VYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNH
YLSYQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGSNDYKDDDD
KGGSHHHHHH**
C1BP7 (SEQ ID NO: 7) MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICT
TGKLPVPWPTLVTTFGYGVQCFARYPDHMRQHDFFKSAMPEGYVQERTIF
FKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHN
VYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNH
YLSYQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGSNDYKDDDD
KGGSHHHHHH**
C1BP8 (SEQ ID NO: 8) MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICT
TGKLPVPWPTLVTTFGYGVQCFARYPDHMRQHDFFKSAMPEGYVQERTIF
FKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHN
VYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNH
YLSYQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGSNDYKDDDD
KGGSHHHHHH**
C1BP9 (SEQ ID NO: 9) MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICT
TGKLPVPWPTLVTTFGYGVQCFARYPDHMRQHDFFKSAMPEGYVQERTIF
FKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHN
VYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNH
YLSYQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGSNDYKDDDD
KGGSHHHHHH**
C1BP10 (SEQ ID NO: 10) MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICT
TGKLPVPWPTLVTTFGYGVQCFARYPDHMRQHDFFKSAMPEGYVQERTIF
FKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHN
VYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNH
YLSYQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGSNDYKDDDD
KGGSHHHHHH**
C1BP11 (SEQ ID NO: 11) MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICT

TGKLPVPWPTLVTTFGYGVQCFARYPDHMRQHDFFKSAMPEGYVQERTIF
FKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHN
VYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNH
YLSYQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGSNDYKDDDD
KGGSHHHHHH**
C1BP12 (SEQ ID NO: 12) MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICT
TGKLPVPWPTLVTTFGYGVQCFARYPDHMRQHDFFKSAMPEGYVQERTIF
FKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHN
VYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNH
YLSYQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGSNDYKDDDD
KGGSHHHHHH**
C1BP13 (SEQ ID NO: 13) MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICT
TGKLPVPWPTLVTTFGYGVQCFARYPDHMRQHDFFKSAMPEGYVQERTIF
FKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHN
VYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNH
YLSYQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGSNDYKDDDD
KGGSHHHHHH**
C1BP14(SEQ II) NO: 14) MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICT
TGKLPVPWPTLVTTFGYGVQCFARYPDHMRQHDFFKSAMPEGYVQERTIF
FKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHN
VYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNH
YLSYQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGSNDYKDDDD
KGGSHHHHHH**
Glucose-Galactose Binding Protein Fusions ttGBP.11CØbZif (SEQ ID NO: 15) MKQLNIGVAICKFDDTFMTGVRNAMTAEAQGKAKLNMVDSQNSQPTQNDQ
VDLFITKKMNALAINPVDRTAAGTIIDKAKQANIPVVFFNREPLPEDMKK
WDKVYYVGAKAEQSGILQGQIMADYWKAHPEADKNHDGVMQYVMLMGQPG
HQDAILRTQYSIQTVKDAGIKVQELAKDYANWDRVTAHDKMAAWLSSFGD
KIEAVFANNDDMALGAIEALKSAGYFTGNKYIPVVGVDATAPGIQAIKDG
TLLGTVLNDAKNQAKATFNIAYELAQGITPTKDNIGYDITDGKYVWIPYK
KITKDNISDAEQGGSGGSTGEKPYKCPECGKSFSRSGGSHHHHHH**
ttGBP.17CØbZif (SEQ II) NO: 16) MKQLNIGVAIYKFDDTCMTGVRNAMTAEAQGKAKLNMVDSQNSQPTQNDQ
VDLFITKKMNALAINPVDRTAAGTIIDKAKQANIPVVFFNREPLPEDMKK
WDKVYYVGAKAEQSGILQGQIMADYWKAHPEADKNHDGVMQYVMLMGQPG
HQDAILRTQYSIQTVKDAGIKVQELAKDYANWDRVTAHDKMAAWLSSFGD
KIEAVFANNDDMALGAIEALKSAGYFTGNKYIPVVGVDATAPGIQAIKDG
TLLGTVLNDAKNQAKATFNIAYELAQGITPTKDNIGYDITDGKYVWIPYK
KITKDNISDAEQGGSGGSTGEKPYKCPECGKSFSRSGGSHHHHHH**

Egi BHHHHEISODbaVUSIMIXIDDIAdIMAANDUL
IaApimaxianobviaAvimaivxvbxxvaminipTunax[vtlloavi VGADAAdIANNWAADVSNIVMVMVIAIWINNVJAVBDICMSS1AWVIAIN
CEEIVIAINIMNIVAGNIFIHbANIDIAINAIbISAbilflIVCOHDdbDIATINAANN St' AouHKxava=dHvxmAavv\abotrnoSbHVNVDAAAA)DaAVXNIAICEdldMIN
aannamvbxvxlmunvviwanamirwmAiminaianbambiabsmbs GAIAININVND6VHVBAIVNIIADENDMICHNAIVADINioNSDOVINVGIMI
lbaNgIVDANIVIVAHON)LITITIAIDIRDAMIVIDdNbUININVAEIND6 AHCIVIarlIdVIIAINDaDDAOVAUCINIIVDCIVNIAGICLISCRIEIHEINCESIAI 0.17 ( I Z :ON 0:11 bas) C'DL, UdEEDIrckl, HHHHEIFISDOVINVCaMlbaNgIVDANIVIVA
HoNalr-rualmlloxxavioambau=rDwArniptuaavlaaimvunix Dappmaymaaniwosavrinaialsaarnilixassoobavasimaxux sc NAdIMAANDULICIADINGXIALLID6V1HAVINILVNV6NNWIWIAIDTIL
DUX[VbIDdIVIVGADAAdIANNDLIADVSNIVMVMVIAIVCINNVJAVBDI
(MIS SlAWVIAINCEEIVIAIMMNIVAGNIFIHbANIDIAINAIbI sAbilnivabH
pabovvimutnninosaHmxsavaaavxmAsavw[botinosbavxvonAAAxam muniaa=nammaannamvbxvxlmunvviwanaminvmnixxunan oc boalloid6SN6SCIAIAININVND6VHVBAIVNUADENDMICLINAIVADINitYNIAI
(CIZ :ON 0:11 bas) X-11.C.DLI.JEDU
**HHHHEIFISDOSIISJSNDDHJDNA=INHDISDDSOD6HVGSINCULDI
NAdIMAANDULKIADINGXIALLID6V1HAVINILVNV6NNWIWIAIDTIL SZ
DCDIPAnDdIVIVGADAAdIANNDLIADVSNIVMVMVIAKKINNVJAVBDI
CMS glAWVIAINCEEIVIAWDNIVAGNIFIHbANIDIAINAIbIsAbilnivabH
pabovvimutnninommxsavaaavxmAsavw[botinosbavxvonAAAxam muniaa=nammaannamvbxvxlmunvviwanaminvmnixxunan bambiabsmbsamAthrixvxotwavEnwmunonnaLaamuvnowitYmni oz (61 :ON sm bas) J!Zcl. DZ 8 I 'clEEDU
**HHHHEIFISDOSIISJSNDDHJDNA=INHDISDDSOD6HVGSINCULDI
NAdIMAANDULKIADINGXIALLID6V1HAVINILVNV6NNWIWIAIDTIL
DUX[VbIDdIVIVGADAAdIANNDLIADVSNIVMVMVIAKKINNVJAVBDI
(MIS SlAWVIAINCEEIVIAIMMNIVAGNIFIHbANIDIAINAIbISAbITIPAItO
pabovvimutnninommixsavaaavxmAsavw[botinosbavxvonAAAxam muniaa=nammaannamvbxvxlmunvviwanaminvmnixxunan bambiabsmbsamAthrixvxotwavEnwmunonnaLaamuvnowitYmni (81 :ON sm bas)PZTODISI'dEEDII 0 **HHHHEIFISDOSIISJSNDDHJDNA=INHDISDDSOD6HVGSINCULDI
NAdIMAANDULKIADINGXLIED6VIHAVINILVNVoNNVCIN1AIDTIL
DUX[VbIDdIVIVGADAAdIANNDLIADVSNIVMVMVIAKKINNVJAVBDI
(MIS SlAWVIAINCEEIVIAIMMNIVAGNIFIHbANIDIAINAIbI SAbilflIVG6H
pabovvimutnninommixsavaaHvxmAsavw[botinosbaDxvonAAAxam muniaa=nammaannamvbxvxlmunvviwanaminvmnixxunan bambiabsmbsamAthrnivxotwavEnwmunonnuiaamuvnowitYmni (LT :om(JJ bas)PZTODI I I'dEEDU
8S6Z90/910ZSI1/134:1 ZI6L80/LIOZ OM

ttGBP.182C.2.Trx (SEQ ID NO: 22) MKQLNIGVAIYKFDDTFMTGVRNAMTAEAQGKAKLNMVDSQNSQPTQNDQ
VDLFITKKMNALAINPVDRTAAGTBDKAKQANIPVVFFNKEPLPEDMKK
WDKVYYVGAKAEQSGILQGQIMADYWKAHPEADKNHDGVMQYVMLMGEPG
HQDAILRTQYSIQTVKDAGIKVQELAKDYANCDRVTAHDKMAAWLSSFGD
KIEAVFANNDDMALGAIEALKSAGYFTGNKYIPVVGVDATAPGIQAIKDG
TLLGTVLNDAKNQAKATFNIAYELAQGITPTKDNIGYDITDGKYVWIPYK
KITKDNISDAEQGGSSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPC
KMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGE
VAATKVGALSKGQLKEFLDANLAGGSHHHHHH
Trx.ttGBP.182C.2 (SEQ II) NO: 23) MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEY
QGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQL
KEFLDANLAGGSKQLNIGVAIYKFDDTFMTGVRNAMTAEAQGKAKLNMVD
SQNSQPTQNDQVDLFITKKMNALAINPVDRTAAGTIIDKAKQANIPVVFF
NKEPLPEDMKKWDKVYYVGAKAEQSGILQGQIMADYWKAHPEADKNHDGV
MQYVMLMGEPGHQDAILRTQYSIQTVKDAGIKVQELAKDYANCDRVTAHD
KMAAWLSSFGDKIEAVFANNDDMALGAIEALKSAGYFTGNKYIPVVGVDA
TAPGIQAIKDGTLLGTVLNDAKNQAKATFNIAYELAQGITPTKDNIGYDI
TDGKYVWIPYKKITKDNISDAEQGGSHHHHHH
Adaptor Proteins Adaptor0 (SEQ II) NO: 24) MSAKIIHLTDDSFDTDVLKADGAILVAFWAEWCGPCKMIAPILDEIADEY
QGKLTVAMLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQL
KEFLDANLAGGSHHHHHH***
Adaptor1.0 (SEQ II) NO: 25) MSAKIIHLTDDSFDTDVLKADGAILVAFWAEWCGPCKMIAPILDEIADEY
QGKLTVAMLNIDQNPGTAPKYGICGIPTLLLFKNGEVAATKVGALSKGQL
KEFLDANLAGGSHHHHHH***
Adaptor2.0a (SEQ II) NO: 26) MSAKIIHLTDDSFDTDVLKADGAILVAFWAEWCGPCKMIAPILDEIADEY
QGKLTVAMLNIDQNPGTAPKYGICGIPTLLLFKNGEVAATKVGALSKGQL
KEFLDANLAGGSHHHHHH***
Adaptor2.0b (SEQ II) NO: 27) MSAKIIHLTDDSFDTDVLKADGAILVAFWAEWCGPCKMIAPILDEIADEY
QGKLTVAMLNIDQNPGTAPKYGICGIPTLLLFKNGEVAATKVGALSKGQL
KEFLDANLAGGSHHHHHH***
Adaptor3.0 (SEQ II) NO: 28) MSAKIIHLTDCSFDTDVLKADGAILVAFWAEWCGPCKMIAPILDEIADEY
QGKLTVAMLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQL
KEFLDANLAGGSHHHHHH***
Adaptor4.0 (SEQ II) NO: 29) MSAKIIHLTDDSFDCDVLKADGAILVAFWAEWCGPCKMIAPILDEIADEY
QGKLTVAMLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQL
KEFLDANLAGGSHHHHHH***
Adaptor5.0 (SEQ ID NO: 30) MSAKIIHLTDDSFDTDVLKACGAILVAFWAEWCGPCKMIAPILDEIADEY
QGKLTVAMLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQL
KEFLDANLAGGSHHHHHH***
Adaptor6.0 (SEQ II) NO: 31) MSAKIIHLTDDSFDTDVLKADGAILVAFWAECCGPCKMIAPILDEIADEY
QGKLTVAMLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQL
KEFLDANLAGGSHHHHHH***
Adaptor7.0 (SEQ II) NO: 32) MSAKIIHLTDDSFDTDVLKADGAILVAFWAEWCGPCKCIAPILDEIADEY
QGKLTVAMLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQL
KEFLDANLAGGSHHHHHH***
Adaptor8.0 (SEQ II) NO: 33) MSAKIIHLTDDSFDTDVLKADGAILVAFWAEWCGPCKMIAPILDCIADEY
QGKLTVAMLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQL
KEFLDANLAGGSHHHHHH***
Adaptor9.0(SEQ II) NO: 34) MSAKIIHLTDDSFDTDVLKADGAILVAFWAEWCGPCKMIAPILDEIADEY
QGCLTVAMLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQL
KEFLDANLAGGSHHHHHH***
Adaptor10.0 (SEQ II) NO: 35) MSAKIIHLTDDSFDTDVLKADGAILVAFWAEWCGPCKMIAPILDEIADEY
QGKLTVAMLNIDCNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQL
KEFLDANLAGGSHHHHHH***
Adaptor11.0 (SEQ II) NO: 36) MSAKIIHLTDDSFDTDVLKADGAILVAFWAEWCGPCKMIAPILDEIADEY
QGKLTVAMLNIDQNPGTAPKCGIRGIPTLLLFKNGEVAATKVGALSKGQL
KEFLDANLAGGSHHHHHH***
Adaptor12.0 (SEQ II) NO: 37) MSAKIIHLTDDSFDTDVLKADGAILVAFWAEWCGPCKMIAPILDEIADEY
QGKLTVAMLNIDQNPGTAPKYGIRGIPTLLLFKNGECAATKVGALSKGQL
KEFLDANLAGGSHHHHHH***
Adaptor13.0 (SEQ II) NO: 38) MSAKIIHLTDDSFDTDVLKADGAILVAFWAEWCGPCKMIAPILDEIADEY
QGKLTVAMLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKCGALSKGQL
KEFLDANLAGGSHHHHHH***

Adaptor14.0 (SEQ ID NO: 39) MSAKIIHLTDDSFDTDVLKADGAILVAFWAEWCGPCKMIAPILDEIADEY
QGKLTVAMLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSCGQL
KEFLDANLAGGSHHHHHH***
Adaptor15.0 (SEQ ID NO: 40) MSAKIIHLTDDSFDTDVLKADGAILVAFWAEWCGPCKMIAPILDEIADEY
QGKLTVAMLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGCL
KEFLDANLAGGSHHHHHH***
Adaptor16.0 (SEQ ID NO: 41) MSAKIIHLTDDSFDTDVLKADGAILVAFWAEWCGPCKMIAPILDEIADEY
QGKLTVAMLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQL
KEFLDCNLAGGSHHHHHH***
EXAMPLES
Examples are provided below to facilitate a more complete understanding of the invention. The following examples illustrate the exemplary modes of making and practicing the invention. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only, since alternative methods can be utilized to obtain similar results.
EXAMPLE 1: SENSING CHLORIDE USING ngmFRET EFFECTS ENGINEERED
INTO YELLOW FLUORESCENT PROTEIN.
As the first, simplest, example of ngmFRET, we use YFP, a mutant of Green Fluorescent Protein (GFP) that contains a serendipitous chloride-binding site located next to its fluorophore (Wachter 1999, Jayaraman 2000, Kuner 2000). Chloride binding elicits a monochromatic response of the fluorophore, quenching its fluorescence with increasing chloride concentrations (by itself and regardless of the presence of any other fluorophore/parter). Binding is not accompanied by a protein conformational change (Grimley et al. 2013), therefore clearly ruling out any distance-dependent effects on energy transfer, which limits the number of possible photophysical models.
The YFP fluorophore is formed by an autocatalytic cyclization of a tripeptide located in an interior a helix (Zimmer 2002). It exists in four possible states (anionic, cationic, zwitterionic, and neutral), of which only the anionic fluorophore is fluorescent(Elsliger 1999) (FIG. 6). Binding of chloride at a site located adjacent to the fluorophore destabilizes the anionic form, raising the excited state pK: resulting in protonation of the fluorophore and quenching of its fluorescence emission intensity(Grimley et al. 2013). The YFP
fluorescence emission spectrum changes intensity but not shape in response to chloride binding (monochromatic response).
We have set up an ngmFRET system in which the fluorophore functions as a directly responsive acceptor, with an indirectly responsive donor site-specifically coupled on the YFP
surface. In this system both donor and acceptor intensities change in response to chloride binding. Our analysis has shown that in the absence of inter-fluorophore distance changes, as is the case for these YFP conjugates, the emission intensity of the indirectly responsive donor can change only if the efficiency of resonance energy transfer from the donor to the directly responsive acceptor changes as a consequence of alterations in the degree of spectral overlap due to ligand-mediated shifts in the excitation spectrum of the directly responsive acceptor.
To construct this ngmFRET system, we used the laYFP variant (Grimley et al.

(FIG. 7), which binds chloride at a site ¨14 A from the YFP phenolic hydoxyl with affinity of ¨160 mM at neutral pH. A parent construct (C1BP0) was designed for heterologous expression in Escherichia coli by optimizing the nucleotide sequence of the open reading frame (Allert, Cox and Hellinga 2010). Thirteen single cysteine mutants (C1BP
2-14) were designed for attachment of the extrinsic indirectly responsive donor fluorophore, located in an annulus around the main YFP (3-barrel and at both ends of the barrel (FIG.
8).
Acrylodan (Prendergast 1983) and Pacific Blue(Sun 1998) were each attached to purified mutant proteins. In these conjugates both donor and acceptor intensities changed in response to chloride and pH, and the ratio of acceptor to donor intensities decreases (FIG. 9;
Table 2). This can happen only if the directly responsive acceptor undergoes dipole switching such that its absorption spectrum undergoes a bathochromic shift and alters the energy transfer coupling factor, thereby decreasing energy transfer efficiency and increasing the intensity of the indirectly responsive donor (equation 16). Chloride binding decreases the fluorescence of the unlabeled YFP, indicating that increased directly responsive acceptor quenching also plays a role in the photophysics of this ngmFRET system. This pattern corresponds to model a+ in Table 1.

Table 2. Chloride affinities and pKa values of semisynthetic YFP chloride sensors'.
Acrylodanb Pacific Blue Kd(C1)(mM) Kd(C1)(mM) Name Mutation Location aPPKd fru% pKa aPPKd iraeKd PKa C1BP2 E17C 01 308 52 5.8 30 60 5.6 C1BP3 E32C 02 19 46 5.8 76 126 5.8 C1BP4 T43C 03 41 129 5.3 96 136 4.4 C1BP5 E95C 134 37 77 5.7 220 340 <4 C1BP6 R109C 135 77 106 5.4 208 545 <4 C1BP7 R122C 06 35 65 5.4 180 360 <4 C1BP8 N149C 137 23 34 5.8 122 106 4.4 C1BP9 N164C 08 26 70 5.3 259 364 <4 C1BP10 Y182C 09 35 88 5.2 105 260 <4 C1BP11 Q204C Om 72 55 5.2 310 242 <4 C1BP12 L221C On 40 140 3.9 167 276 <4 C1BP13 H77C End 20 55 5.6 150 290 <4 C1BP14 D173C End 78 110 5.4 217 212 <4 a Determined by fitting ratiometric signal of the intensities measured at k1 and k2 to equations 20-25.
bRatiometry: ki=530 nm, k2=500 nm.
cRatiometry: k 1=530 nm, k2=455 nm.
The collection of thirteen YFP conjugates (Table 2) illustrates that the quantitative details of their ngmFRET behavior have profound effects on sensor utility, even if all constructs evince ratiometric responses to chloride binding. The emission peak of Acrylodan is centered at ¨500 nm, whereas Pacific Blue emits at 455 nm. The excitation maximum of YFP is centered at ¨515 nm. The spectral overlap with the YFP acceptor therefore is much greater for Acrylodan than for Pacific Blue donors. Consequently the donor emission intensity is weak in Acrylodan conjugates, as most of their excited state energy is transferred to YFP, whereas the corresponding intensities of Pacific Blue donors are much stronger.
Furthermore, the Pacific Blue peak is more responsive than Acrylodan, as small shifts in spectral overlap by changes in the directly responsive YFP acceptor have a relatively larger effect on the former than the latter. Even among the Pacific Blue conjugates, there is quantitative difference in the degree of overlap, depending on the attachment position, with concomitant changes in behavior (Table 2). Effective dichromatic chloride sensors therefore are constructed by balancing the degree of spectral overlap such that the donor intensity contributes significantly to the signal.
The response of biosensors based on Langmuir binding isotherms is most sensitive at analyte concentrations that match the apparent Kd value of the protein (Marvin et al. 1997).

The various Acrylodan and Pacific Blue conjugates therefore can be used to measure chloride concentrations over two orders of magnitude, in the 10-1000 mM concentration range. These concentration ranges are relevant to water quality determination, corrosion, and clinical chemistry. The clinical reference range (Burtis 2012) for chloride concentrations is 90-120 mM (normal serum range 97-107 mM). The Pacific Blue conjugates of C1BP3, 6, 8 and 10 exhibit aPPKd values centered around 100 mM Cl" in solutions at pH 7.4 with ionic strengths of ¨150 mEq (conditions that match that of serum(Burtis 2012)), and therefore are good candidates for use in point of care (POC) technology. The pKa values of these variants are less than 4; fluctuations in proton concentrations around pH 7.4 therefore will not interfere with the functioning of these biosensors, which is an important consideration in the development of chloride detection technologies.
EXAMPLE 2: SENSING GLUCOSE USING ngmFRET EFFECTS ENGINEERED
INTO A THERMOSTABLE GLUCOSE, BINDING PROTEIN.
As a second, more complex example we use a glucose-binding protein in which the directly responsive fluorophore is site-specifically coupled to a cysteine mutant located in the binding pocket of the protein. The indirectly responsive partner is placed either on a C-terminal PZif or N-terminal thioredoxin fusion, using the multiple thiol-labeling strategy described above. We provide examples of directly responsive donors and acceptors.
A thermostable homolog of Escherichia colt glucose-galactose binding protein, ecGGBP (Vyas, Vyas and Quiocho 1988), was identified in the thermophilic bacterium Thermoanaerobacter thermosaccharolyticum. Additional non-limiting examples of glucose-galactose binding proteins, as well as other ligand-binding proteins, are disclosed herein.
Additionally, non-limiting examples of glucose-galactose binding proteins are described in PCT International Patent Application No. PCT/US2016/050297, filed September 2, 2016.
This homolog, ttGGBP, is a member of the bacterial periplasmic-binding protein (PBP) superfamily, which has provided several ligand-responsive reagentless fluorescent sensors (Grunewald 2014). The structure of PBPs comprises two domains connected by a hinge (Bemtsson et al. 2010). Ligand binding shifts a conformational equilibrium via a hinge-bending motion from an ensemble of predominantly "open" states to a "closed"
state in which the bound ligand is enveloped within the interface between the two protein domains.
Using site-specific modification of cysteine mutants, environmentally sensitive fluorophores have been placed strategically to detect these ligand-mediated conformational changes (de Lorimier et al. 2002). The resulting semi-synthetic, fluorescent PBP
conjugates then function as reagentless, fluorescent biosensors for their cognate ligands.
We constructed a series of single cysteine mutants in ttGGBP (FIG. 10) and evaluated the fluorescent responses of Badan and Acrylodan conjugates at these positions. Three of the seventeen positions exhibited dichromatic responses with one or both of these fluorophores (Table 3). The best ratiometric responses were seen for Badan and Acrylodan attached to Fl7C and W182C, respectively. A series of additional mutations were introduced within these two cysteine mutants to tune their glucose affinities (Table 4).
Finally, we tested the responses of a variety of different fluorophores (FIG. 11) at these two positions (Table 5).
Most of these fluorophores evinced monochromatic responses, with the exception of the W182C=IAEDANS conjugate which exhibited a small change in emission spectrum shape upon binding glucose. These singly labeled conjugates form a dataset of directly responsive fluorophore from which examples were drawn to construct glucose-sensing ngmFRET
systems.
Table 3. Glucose response of Acrylodan and Badan conjugates in a cysteine scan of the ttGGBP scaffold.
Mutation Class' Shapeb Conjugate' aP 7' Emission Kdd,e (K) wavelen,gth (nm) (Inn aPPICa trueKd Y11C P m A 351 511 470 0.12 0.16 d B 349 492 528 0.28 0.24 T16C P m A 351 519 460 8.8 12 m B 349 nb nb F17C e d A 349 482 542 0.08 0.06 d B 346 467 491 0.087 0.26 N42C P A 350 nb nb B 324- - nb nb V67C P A 350- - nb nb B 322- - nb nb R91C e m A 349 491 540 0.18 0.17 B 348 nbd nbd E92C P A 350- - nb nb B 346- - nb nb AMC P m A 350 515 550 0.19d 0.11d m/d B 348 523 550 0.64 0.55 Q148C p A 351 nb nb B 349- - nb nb H151C e m A 351 511 489 0.012 0.025 m B 348 523 550 0.018d 0.027d Q152C P A 351 - - nbd nbd B 349 - - nb nb N181C P A 350 - - nb nb B 349 - - nb nb W182C e d A 347 479 526 2.3 2.3 m B 347 515 550 27 19 D183C P A 348 - - nbd nbd B 348 - - nb nb L257C a A 352 - - nb nb B 348 - - nb nb D259C a A 349 - - nb nb B 347 - - nb nb K300C a A 349 - - nb nb B 348 - - nb nb aa, allosteric; e, endosteric; p, peristeric.
bm, monochromatic; d, dichromatic (i.e. spectral shape change).
A, Acrylodan; B, Badan.
dnoisy data and or bad fit.
enb; no binding, nd; not determined.
Table 4. Responses of mutant ttGGBP17C and ttGGBP182C conjugates'.
Emission aP T. wavelength Glucosed Galactosed Protein Mutation Classb Conjugate' (nm) S
(K) 2,1 X2 aPPKd tniaKd tin%
(n1M) (n1M) (nlivi) ttGGBP17C B 346 467 519 0.10 0.15 0.19 1.3 99 A 349 487 515 0.08 0.09 3.8 43 ttGGBP17C.1 R91K,Q148E 2 B 463 515 0.6 0.8 0.4 0.46 ttGGBP17C.2 R69P,Q152P 2 B 350 479 523 8.6 10.8 5.2 0.48 99 99 2 A 492 515 15 18 5.0 0.28 ttGGBP17C.3 T16N,D211A 2 B 346 471 531 3.4 4.1 2.5 0.61 99 99 2 A 495 529 1.4 1.5 0.53 0.35 ttGGBP17C.4 H151Q 1 B 347 511 457 16 6.4 14 2.2 14 0.29 ttGGBP17C.5 D15A 1 B 345 487 530 16 16 4.2 0.26 99 99 1 A 348 483 498 6 6 2.4 0.41 ttGGBP17C.6 DISE 1 B 346 467 525 3.3 3.6 3.6e 1 ttGGBP17C.7 D15N 1 B 347 483 515 0.75 1.0 0.64 0.64 99 99 1 A 348 483 515 0.3 0.3 0.3 0.91 ttGGBP17C.8 T16N 2 A 348 487 529 0.61 0.60 0.26 0.43 ttGGBP17C.9 T16S 2 A 487 520 0.2 0.20 nd ttGGBP17C.10 G20A 3 A 351 487 520 0.4 0.4 nd ttGGBP17C.11 T240A 3 A 348 487 500 0.04 0.04 nd ttGGBP17C.19 N258D 3 B 344 nbe nbe nbe ttGGBP17C.20 N258S 3 B 344 nbe nbe nbe ttGGBP17C.21 N258A 3 B 345 532 494 61 69 nb ttGGBP17C.22 A260N 3 B 343 523 492 12 15 530e ttGGBP17C.23 A260Q 3 B 344 527 490 15 18 nbc ttGGBP17C.24 A260R 3 B 344 490 515 1.8 1.7 29 17 ttGGBP17C.25 A260K 3 B 515 493 3.2 3.7 nbe ttGGBP17C.26 A260W 3 B 346 523 509 0.9 0.9 14 16 ttGGBP17C.27 A260F 3 B 346 523 490 0.2 0.3 8.8 33 ttGGBP17C.28 A260Y 3 B 346 523 494 0.06 0.07 2.6 37 ttGGBP17C.29 A2605 3 B 343 527 496 2.1 2.2 250 114 ttGGBP182C A 347 472 535 2.2 2.3 3.3 1.4 ttGGBP182C.2.0f R91K Q148E 2,3 A 346 475 545 4.5 6.0 18.5 3.1 ttGGBP182C.2.1g A1545 1 A 328 480 552 3.0 4.1 13.1 3.2 ttGGBP182C.2.3g A154N 1 A 346 470 537 16.3 19.0 207 10.9 ttGGBP182C.2.4g A154M 1 A 346 472 540 0.4 0.4 4.5 11.2 ttGGBP182C.2.5g H151Q 1 A 346 477 542 10.8 13.0 20.3 1.6 ttGGBP182C.2.6g H151N 1 A 347 475 537 19.2 17.9 52.6 2.9 ttGGBP182C.2.7g H151F 1 A 346 475 545 121 124 ttGGBP182C.2.8g D15N 1 A 343 474 542 179 209 372 1.8 ttGGBP182C.2.9g A154F 1 A 345 512 477 0.4 0.3 1.6 4.0 ttGGBP182C.3f R91K 2 A 346 494 459 1.8 0.9 3.1 3.4 ttGGBP182C.4f Q148E 2 A 347 475 542 0.6 0.8 6.5 8.1 ttGGBP182C.5f R69P Q152P 2 A 349 477 535 7.5 8.8 7.4 0.8 ttGGBP182C.6f T16N D211A 2 A 345 472 530 21.6 27.6 81 2.9 ttGGBP182C.7f R91K Q1485 2,3 A 346 478 550 0.3 0.4 1.1 3.7 ttGGBP182C.8f 2,3 A 346 475 545 28.7 44.2 256 5.8 ttGGBP182C.9i D15N 1 A 343 475 540 75.1 76.4 282 3.7 'Measured on the Nanodrop at room temperature. kina,, is the wavelength corresponding to the maximum emission intensity. Optimal ratiometry wavelengths are determined according to the analysis described in Materials and Methods. The tmeKd is determined from monochromatic titration curves; aPPKd from dichromatic ratiometry (equations 20-25).
Average relative error in the trueKd values is 5%, in the aPPKd values, 1%. S
is the selectivity between glucose and galactose, S=mleKd(galactose)/ mieKd(glucose); S>l, selective for glucose.
pl, PCS; 2, inter-domain interaction; 3, contact between protein and fluorophore CA, acrylodan; B, badan.
dnb, no bonding; nd, not determined.
eNoisy data or bad fit.
'Additional mutation constructed in ttGGBP182C.
gAdditional mutation constructed in ttGGBP182C.2Ø
Table 5. Responses of fluorophores conjugated to F17C and W182C mutants of ttGGBP'.
b Xex alxiXmax aPoimax satxmax satimax true-d Position Fluorophore (nm)` (nm) (AU x1000) (nm) (AU x1000) (mNI) 17C Acrylodan 391 487 15.9 487 20.0 0.2 Badan 391 519 12.8 467 52.6 0.1 5-IAF 491 523 64.3 523 70.6 61.3 Oregon green 496 523 101.9 523 91.7 -CPM 384 471 78.5 467 90.9 17.0 IANBD 478 531 15.3 535 19.8 11.8 IAEDANS 336 467 12.5 467 14.4 -Pacific Blue 410 451 28.6 455 108.6 60.7 182C Acrylodan 391 479 55.0 515 17.2 6.0 Badan 391 515 16.8 515 11.9 64.9 5-IAF 491 519 255.0 519 453.2 5.0 6-IAF 491 513 50.4 513 63.1 750 Oregon green 496 519 78.2 519 186.2 20.0 CPM 384 479 49.5 483 40.3 6.8 IANBD 478 543 29.1 547 12.5 210 IAEDANS 336 487 3.7 483 7.9 0.2 Pacific Blue 410 455 115.0 455 119.4 -BODIPY 499 499 519 40.1 515 80.2 30.7 BODIPY 507 507 531 32.9 535 31.4 11.0 Alexa 488 495 519 211.6 519 182.2 -Alexa 532 532 551 63.0 551 61.0 -Alexa 546 546 571 152.1 571 150.2 -Texas Red 595 611 23.3 611 23.5 -Cy5 646 663 19.2 663 23.3 210 PyMPO 415 555 3.7 559 4.2 20.6 akex, preferred excitation wavelength (from supplier); aP 2k.max, observed maximum emission wavelength of the apo-protein; aP /max, observed intensity at ank.max; satA,, , max, observed maximum emission wavelength of the glucose complex; sat/max, observed intensity at satkmax;
true/Cd, affinity determined from fit of equations 20-25 to the monochromatic emission intensities. Emission spectra were measured on the Nanodrop3300, using -10 iiM
protein.
The observed absolute emission intensities are a rough guide to the relative brightness of the conjugate, because the protein concentration was approximately the same for each experiment.
bAbbreviations, chemical names and supplier catalogue numbers as follows:
Acrylodan (A433); Badan (B6057); 5-IAF (130451); Oregon Green 488 (06034); CPM (D346);
IANBD
(D2004); IAEDANS (114); Pacific Blue (P30506); BODIPY 499 (D20350); BODIPY 507 (D6004); 6-IAF (130452); Alexa 488 (A10254); Alexa 532 (A10255); Alexa 546 (A10258);
Texas Red (T6008); PyMPO (M6026) from Life Technologies and Cy5 (13080) from Lumiprobe.
eThe Nanodrop3300 fixed wavelength LED that most closely matched kex was used (see Materials and Methods).
Conversion of monochromatic to dichromatic responses.
Case 1: Directly responsive donor, indirectly responsive acceptor; pure quenching (model cr 09) The singly labeled Pacific Blue conjugate at F 17C exhibits a strong increase in emission intensity without significant shifts in emission wavelength maxima in response to glucose (FIG. 12A), suggesting that the changes are due primarily to a decrease in the non-radiative decay rate of its excited state in the glucose complex. In the doubly labeled Fl7C Pacific Blue f3Zif 5-IAF fusion the emission intensities of both the directly responsive donor and indirectly responsive acceptor peaks increase with glucose addition (FIG. 12B).
This correlated increase of both donor and acceptor emission intensities is inconsistent with effects confined to changes in the energy transfer coupling factor, 0. The lack of observable changes in the emission spectrum, indicates that the ngmFRET is modulated only by differences in the non-radiative decay rate on the Pacific Blue directly responsive donor in the ligand-free and ¨bound protein (model d 0 , Table 1). In model JO, modulation of the indirectly responsive acceptor emission intensity are due to changes in the resonance transfer rate which increases as a consequence of decreased the donor non-radiative decay rate (by itself and regardless of the presence of any other fluorophore/parter).
The PZif fusion domain is located at the back of the ttGGBP hinge region, close to the attachment point of the directly responsive fluorophore at position Fl7C or W182C (FIG.
13), forming an ensemble of indirectly responsive fluorophore conformations.
In this arrangement, the ligand-mediated protein conformational change that converts the open to the closed state is unlikely to change the average distance or orientation between the single directly responsive fluorophore conformation and the ensemble of indirectly responsive fluorophores. The behavior of the F17C= Pacific Blue f3Zif 5-IAF ngmFRET pair is consistent with this interpretation. Distance contributions therefore can be ruled in the interpretation of the intensity change pattern.
Singly labeled Acrylodan attached to position 111C also exhibits a monochromatic increase in emission intensity in response to glucose binding. When paired as a directly responsive donor with f3Zif Alexa532 as the indirectly responsive acceptor, this monochromatic signal is converted to a dichromatic one (Table 7). As with the F17C=Pacific Blue ¨ f3Zif IAF example described above, donor and acceptor emission intensities increase unequally upon binding glucose, enabling ratiometry, and indicating that the JO model is operational in this construct as well. By contrast, glucose binding evinces only a monochromatic signal in the 111C=Acrylodan - f3Zif IAF system. In this construct the wavelengths of the Acrylodan donor and IAF acceptor emission peaks are so close together that their changes in either cannot be resolved independently. The same effects are observed for the Badan conjugate attached to position 151C both singly labeled and partnered with f3Zif Alexa532 and f3Zif IAF (Table 7). These observations illustrate the importance of carefully tuning donor-acceptor optical properties in order to obtain dichromatic signals, even if ngmFRET between the two partners can be established.

Case 2: Directly responsive donor, indirectly responsive acceptor; pure quenching (model d+ 0 ) In contrast to the Badan conjugate described above, at positon 151C the singly labeled Acrylodan conjugate exhibits a monochromatic emission intensity decrease. When partnered with an indirectly responsive gif Alexa532 acceptor, both donor and acceptor intensities decrease (Table 6). This behavior is consistent with the pure quenching (change in the ratio of radiative to non-radiative emission rates) model di-0 (Table 1). For the reasons described above, the 151C=Acrylodan - f3Zif IAF system evinces a monochromatic response.
Case 3: Indirectly responsive donor, directly responsive acceptor: combined spectral shift and quenching (model a- 0).
The fluorescence intensities of W182C conjugates singly labeled with closely related members of the fluorescein family, 5-IAF and Oregon Green (FIG. 11D and E), increase in response to glucose binding (Table 5). This observation suggests that the glucose complexes of these two conjugates exhibit a significant decrease in the relative rates of non-radiative decay.
These conjugates were paired as directly responsive acceptors with Pacific Blue as their indirectly responsive donor. The indirectly responsive donor emission intensity increased in concert with that of the directly responsive acceptor in both ngmFRET pairs (FIG. 14). As described for YFP in the first example, such a pattern is inconsistent with pure geometrical effects. The "feedback" to the indirectly responsive donor indicates that ngmFRET efficiency has diminished by a bathochromic shift in the directly responsive acceptor excitation spectrum, resulting in increased donor emission. This can occur only if the directly responsive acceptor switches dipoles, consistent with the presence of multiple excited states in Fluorescein and Oregon Green (Martin 1975). The increase in intensity of the singly labeled conjugates indicates that quenching decreases upon glucose binding. The modulation mechanism therefore combines quenching and ngmFRET coupling effects: model a4 (Table 1).
In both systems, the fluorescent response pattern changes with increased glucose concentrations, shifting from an increase (Phase I) to a decrease (Phase II) in the QD/QA ratio (Fig. 14), indicating that there are two types of glucose responses. This phenomenon strongly suggests the presence of a second, much weaker affinity glucose-binding site that influences the environment of the directly responsive acceptor. The intensity of the indirectly responsive donor continues to increase with glucose concentration in Phase II, indicating a further decrease in 0 with a concomitant bathochromic shift of the acceptor absorption spectrum. The pattern of changes in the QD/QA ratio is quantitatively dependent on the change in magnitudes of a and 0 in the ci 0" model (Table 1). Accordingly, the change in the direction of the ratiometric signal indicates that the relative sizes of the change in 0 and a are different in Phases I and II.
Table 6. Glucose affinities of ngmFRET systems in Yl1C, A111C, and H151C
ttGGBP
conjugates.
Emission wavelengths (nm) Affinity (mM)a Conjugate Xi X2 aPPKd iraeKd 11C=Badan gif5-IAF 480 565 0.16 0.16 110Badan gifAlexa532 480 565 0.14 0.14 1110Acry1odan gifAlexa532 495 575 0.015 0.012 1510Acrylodan gifA1exa532 520 575 0.28 0.11 1510Badan gif=A1exa532 515 580 0.01 0.002 'Determined by fitting ratiometric signal of the intensities measured at Xi and k2 to equations 20-25.
Table 7. Ligand affinities of ngmFRET systems of affinity-tuned mutants within the Fl 7C
and W182C ttGGBP background.
Emission aPPKd (11.1W
wavelengths (nm) Glucose Galactose Conjugate X1 X2 R91,Q148 R91K,Q148E R91,Q148 R91K,Q148E
170Badan gif5-IAF 467 520 0.2 0.2 170Badan gifA1exa532 467 560 0.2 0.2 170Pacific Blue 13ZiF5-1AF 456 520 31 19 1820Acrylodan gif5-IAF 465 520 2.0 4.4 3.7 18.4 1820Acrylodan gifA1exa532 480 549 1.7 3.6 182054AF 13Zif=Pacific Blue 455 520 2.9 9.9 nbb 18200regon Green 13Zif=Pacific Blue 455 520 1.8 11.6 nbb 1820IAEDANS gif5-IAF 465 520 0.1 5.3 'Determined by fitting ratiometric signal of the intensities measured at k1 and k2 to equations 20-25.
bNo binding. Phase II response only.

Improvements of dichromatic responses.
Case 1: enhancing intensities in strong ratiometric signals (models . 1 0- and d* 0 ).
Single Badan and Acrylodan conjugates attached to the F17C and W182C cysteine mutations within the glucose-binding site of ttGGBP exhibit strong dichromatic responses to glucose (FIG. 15). Remarkably, the emission intensity maxima shift in opposite directions at these two positions: glucose binding evinces a bathochromic shift for 182C=Acrylodan, whereas F17C=Badan exhibits a hypsochromic response. Analysis of the emission intensity (FIG. 16) and absorption spectra (FIG. 17) indicated that glucose binding alters the populations of two dominant electronic transitions in both the excited (Si and S2) and ground (Gi and G2) states. These two conjugates therefore switch dipoles in response to glucose binding. In the F 1 7C=Badan hypsochromic response, the dominant excited state electronic transition shifts from Si (green) in the apo-protein, to S2 (blue) in the glucose complex; in the 182C=Acrylodan bathochromic response, the opposite redistribution is observed, and the glucose complex is dominated by the Si (green) excited state. Similarly, in the ground state, the hypsochromic response shift the electronic transitions in the absorbance spectra from a low- (Gi) to a high-energy state (G2); whereas in the bathochromic response the shift is G2¨> Gl .
Doubly-labeled conjugates were prepared, using C-terminal PZif fusions, in which Fl7C=Badan and W182C=Acrylodan function as directly responsive donors and f3Zif 5-IAF or f3Zif Alexa532 as indirectly responsive acceptors (Table 7). These two acceptors have different spectral characteristics and degrees of spectral overlap with their donors.
All four conjugates exhibit significant changes in fluorescence intensities in response to glucose (FIG. 18). These responses can be divided into two glucose concentration phases.
In Phase I which covers the 0-50 mM glucose concentration range, the directly responsive donors exhibited large changes in emission intensities (increases for Fl7C=Badan, decreases for W182C=Acrylodan conjugates), whereas the indirectly responsive acceptors showed only small, but discernable changes. At >50 mM glucose concentrations in Phase II, the intensities of both directly responsive donor and indirectly responsive acceptor peaks increase in concert.
The singly labeled 17C=Badan and 182C=Acrylodan conjugates are good ratiometric sensors. Nevertheless, their incorporation into an ngmFRET system provides additional advantages. First, it extends the wavelength range over which intensities are sufficiently bright to be measured precisely. This is particularly clear for the f3Zif Alexa532 acceptors, which provide bright signals around 560 nm whereas neither the singly labeled Badan nor the Acrylodan constructs are particularly bright at this wavelength (compare FIGS.
15 and 18).
Second, the ngmFRET systems increase the brightness of the sensors over the entire range.
For instance, the glucose complex of the singly labeled 182C=Acrylodan conjugate is approximately 50% less bright compared to the apo-protein (Fig. 15b), whereas these two forms differ by only ¨20% when partnered with f3Zif Alexa532.
In Phase I, both the A1exa532 and the 5-IAF indirectly responsive partners decreased in emission intensity in response to the F17C=Badan directly responsive partner, whereas when partnered with W182C=Acrylodan, they showed an increase in intensity.
This observation is consistent with the two directly responsive partners undergoing dipole switches in the opposite direction: F17C=Badan, green¨*blue (hypsochromic shift;
dim¨*bright; model cr0", Table 1); 182C=Acrylodan, blue¨*green (bathochromic shift;
bright¨*dim; model dff, Table 1).
In Phase II, at glucose concentrations in excess of'-50 mM, the intensities of both donor and acceptor peaks increase in all four conjugates. This is consistent with a second, low-affinity glucose binding site, as observed for the directly responsive 5-IAF and Oregon Green acceptor system described above. Although both donor and acceptor peak intensities increase, the QA/QD decreases, which is inconsistent with only a decrease in donor quenching; a change in ngmFRET is also implicated (model 60.
In addition to using C-terminal PZif fusions, we also attached indirectly responsive ngmFRET partners to N- and C-terminal ecTRX fusions (Table 8). For these experiments, we used the affinity-tuned 17C.3 and 182C.2 variants (Table 4). In all cases, we used A1exa352 as the indirectly responsive ngmFRET partner. Surprisingly, ngmFRET
was not established in either the N- or C-terminal fusions between 182C.2=Acrylodan and ecTRX
(Table 8), nor in the N-terminal ecTRX-17C.3=Badan fusion; only the C-terminal 17C.3=Badan-ecTRX exhibited coupling between the directly responsive donor and indirectly responsive acceptor fluorophores (FIG. 19). The response of this fusion exhibited pattern similar to that observed for its PZif counter-part, indicating that, once established, the ngmFRET mechanism operates in a manner consistent with the character of the directly responsive fluorophore and the logic of the donor-acceptor arrangement. This results also indicates that the nature of the site-specific attachment point of the indirectly responsive partner is critical. For instance, the ecTRX fusions could have placed the acceptor too far away, or in the incorrect relative orientation relative to the donor for efficient ngmFRET.
Table 8. Ligand affinities of ttGGBP-Thioredoxin fusion ngmFRET pairs'.
Glucose (mM) Emission wavelengths (nm) Conjugate 261 k2 appiCd trued Trx=Alexa532-ttGGBP17C.3=Badan 467 543 3.1 3.1 ttGGBP17C.3=Badan-Trx=Alexa532 467 547 3.6 3.7 Trx=Alexa532- 4.7 3.2 ttGGBP182C.2.0=Acrylodan 475 515 ttGGBP182C.2.0=Acrylodan- 5.8 4.3 Trx=A1exa532 475 519 'Determined by fitting ratiometric signal of the intensities measured at k1 and k2 to equations 20-25.
Case 2: enhancing a weak ratiometric signal (model d-q5).
The W182C=IAEDANS-gif 5-IAF (fluorescein) construct is another example of a directly responsive donor- indirectly responsive acceptor ngmFRET pair. The singly labeled W182C=IAEDANS conjugate exhibits a modest hypsochromic shift in response to glucose, which, to a first approximation, involves two electronic transitions (FIG.
20). Gaussian fits revealed that the population of a green state dominates in the apo-protein and exchanges for a shorter wavelength (blue) form in the glucose complex (FIG. 20). This behavior is reminiscent to that of the other naphthalene-based fluorophore, Acrylodan, suggesting that IAEDANS also undergoes a glucose-mediated dipole switch.
The W182C=IAEDANS-gif 5-IAF ngmFRET pair exhibits a concerted increase in the directly responsive IAEDANS donor and indirectly responsive 5-IAF acceptor emission intensities in response to glucose (FIG. 20C). As with the W182C=Acrylodan conjugate, this response also can be divided into two phases, corresponding to high- and low-affinity glucose binding sites (FIG. 20D). In both phases, the response pattern is consistent with donor dipole switching in which a decrease in spectral overlap due to the hypsochromic shift is accompanied by a decrease in non-radiative decay (model chi)", Table 1).
The singly labeled Badan conjugate attached at position 11C undergoes a green¨*blue hypsochromic shift in response to glucose binding with a concomitant overall increase in intensity (Table 3). Partnering this directly responsive donor with a f3Zif IAF or f3Zif Alexa532 indirectly responsive acceptor results in an ngmFRET system that exhibits increases in the emission intensities of both donor and acceptor peaks, and a decrease in their ratio (Table 6), consistent with the d 0" model (Table 1). The wavelength separation of the donor and acceptor peaks is sufficiently large in the 11C=Badan - f3ZiPIAF to obtain a dichromatic signal.
EXAMPLE 3: ENGINEERING RATIOMETRIC SIGNALS USING
MONOCHROMATIC CHEMOSENSORS AND ADAPTOR PROTEINS.
Here we demonstrate that ngmFRET can be extended to convert monochromatic chemosensors to dichromatic responses, by using an "adaptor" protein to pair directly and indirectly responsive fluorophores via site-specific, orthogonal conjugation chemistries. We used the pH-dependent response of Fluorescein (Martin 1975) as an example of a monochromatic chemosensor. Ionization of the phenolic hydroxyl (FIG. 21) has a pKa value of 6.7, and is accompanied by a change in emission intensity, with the dianion having a higher quantum yield than the monoanion. Ionization is accompanied by a bathochromic shift of the absorption and excitation spectra, enabling ratiometric measurements based on absorption or fluorescence excitation, but not emission wavelengths(Lin 2000, Han 2010, Wencel 2014). Fluorescein was paired as the chemoresponsive acceptor with Pacific Blue(Sun 1998) or Acrylodan (Prendergast 1983) as pH-insensitive ngmFRET
donors. The two fluorophores were attached to adaptor proteins constructed out of mutants of Escherichia coli thioredoxin (ecTRX), a member of the thioredoxin protein superfamily(Holmgren 1985, Qi 2005, Yoshioka 2015, Amer 2000), which were engineered to enable orthogonal, site-specific conjugation chemistries at two independently addressable sites.
Two different orthogonal fluorophore attachment chemistries were engineered.
In the first (Adaptor1.0 and 3.0-16.0), the existing ecTRX disulfide was combined with an engineered surface cysteines (FIG. 22). This arrangement enables site-specific attachment of one thiol-reactive fluorophore to the surface, keeping the disulfide oxidized, and a second to the disulfide, following reductive deprotection. Adaptor1.0 is remarkably thermostable, with a Tin value of at least 100 C at neutral pH. In the second adaptor protein (Adaptor2.0), we created a "reduced alphabet" protein that lacks lysines, leaving the N-terminus as the only primary amine. This arrangement enables site-specific attachment of an amine-reactive fluorophore to the adaptor N-terminus, and a second fluorophore to the reduced disulfide.

The thermostability of this protein is more modest at ¨60 C, but sufficient for use in instrumentation.
Construction of adaptor proteins.
Wild-type ecTRX is a 108-residue a/f3 protein that contains a single disulfide linkage within a surface loop(Katti 1990) (FIG. 22). As a starting point for building adaptor proteins (Adaptor0) we introduced several mutations that remove an adventitious Cu(II)-binding site at the N-terminus (D2A) and charged residues buried within the hydrophobic core (D26A, K27M) that tune the redox potential of the disulfide (Ladbury 1993, Hellinga 1992, Langsetmo 1991b, Langsetmo 1991a) (FIG. 23). A hexahistidine purification tag was fused to the C-terminus. In Adaptor1.0 and 3.0-16.0, we further introduced surface cysteine residues. These designs enabled site-specific dual labeling with thiol-reactive probes, the first to the surface thiol, and the second to the disulfide following a reductive deprotection step.
Adaptor2.0a and 2.0b are highly unusual proteins in which we limited the amino acid alphabet to 19 residues, and replaced all remaining nine, surface lysines with arginine (2.0a) or a combination of arginine and glutamine (2.0b). In these designs, thiol-reactive probes are attached at the reduced disulfide, and amine-reactive probes at the N-terminus.
The response of Adaptors 1.0 and 3.0-16.0 conjugates to pH.
Four dually labeled Adaptor1.0 conjugates were prepared, attaching the thiol-reactive Fluorescein derivative 5-IAF to the surface thiol or reduced disulfide, and Acrylodan or Pacific Blue to the corresponding, orthogonally reactive site. All conjugates were illuminated at 365 nm in a Nanodrop3300 spectrofluorimeter, and their emission intensity spectra recorded as a function of pH. All four conjugates exhibited ngmFRET from the indirectly responsive Acrylodan or Pacific Blue donor to the directly responsive Fluorescein acceptor (FIG. 24A-D, Table 9). In all cases, to a first approximation, the intensity of the Fluorescein acceptor decreased, but not the Acrylodan or Pacific Blue donor changed. This pattern of intensity changes in characteristic of a ngmFRET system in which the directly responsive acceptor quenching changes (a+0 , Table 1). The increase of the Fluorescein emission intensity with pH is consistent with the higher quantum yield of the dianion compared to the monoanion.

Table 9. pKa values of conjugates measured by emission and absorbance intensities'.
Fluorescein plC, Adaptor Donor attachment Emission Absorbance 1.0 Acrylodanb R73C 6.2 6.4 Disulfide 6.1 6.6 Pacific Bluee R73C 6.2 6.8 Disulfide 6.2 6.7 2.0a Acrylodanb N-terminus 5.8 6.1 2.0b Acrylodanb N-terminus 5.8 'Determined by fitting ratiometric signal of the intensities measured at Xi and k2 to equations 20-25.
bRatiometry: Absorbance, k1=400 nm and k2=496 nm; Emission, k1=465 nm and k2=520 nm.
cRatiometry: Absorbance, k1=411 nm and k2=496 nm; Emission, k1=455 nm and k2=520 nm.
The choice of fluorophore attachment sites affects the ngmFRET efficiencies between the donors and fluorescein acceptor, as judged by the relative intensity of the donor emission.
Labeling of the disulfide rather than the surface R73C thiol with the donor gives better ngmFRET for either the Acrylodan or Pacific Blue donors. Since the distances are approximately the same in these two arrangements, any differences must be due to changes in dipole-dipole orientation (Valeur 2012, Lakowicz 2006, Clegg 1995, Wu 1994, Cheung 1991). ngmFRET is more in efficient in the Acrylodan than the Pacific Blue conjugates, presumably reflecting difference in spectral overlap. Fluorescein is maximally excited at 490 nm, which is better matched with the maximum emission intensity of planar Acrylodan at ¨500 nm (Allert 2015), than the 450 nm maximum of Pacific Blue.
The absorption spectra of the dually-labeled conjugates shift in response to pH (Fig.
25A-B), with a strong peak at ¨ 500 nm appearing with the formation of the phenolate dianion at high pH value (Martin 1975).
Adaptors 3.0-16.0 were labeled with Acarylodan/Fluorescein and Pacific Blue/Fluorescein pairs (Table 10). All conjugates demonstrated ngmFRET
responses to proton binding.

Table 10. pKa values of Adaptor conjugatesa.
Name Cysteine Fluorophore Fluorophore Emission p/f.
position Cysteine disulfide wavelength (nm) kl 262 aPPO4 fr1ep/4 Ada3.0 D11C Acrylodan 5-IAF 523 460 6.11 6.11 Ada4.0 T15C Acrylodan 5-IAF 523 460 6.23 6.29 Ada5.0 D21C Acrylodan 5-IAF 523 460 6.32 6.36 Ada6.0 W32C Acrylodan 5-IAF 515 460 5.66 5.64 Ada7.0 M38C Acrylodan 5-IAF 515 460 5.84 5.83 Ada8.0 E45C Acrylodan 5-IAF 515 480 6.1 5.93 Ada9.0 K53C Acrylodan 5-IAF 523 460 6.2 6.14 Ada10.0 Q63C Acrylodan 5-IAF 519 465 5.9 5.98 Ada11.0 Y71C Acrylodan 5-IAF 515 460 5.95 5.92 Ada12.0 V87C Acrylodan 5-IAF 523 485 6.22 6.23 Ada13.0 V92C Acrylodan 5-IAF 523 540 6.78b 7.05b Ada14.0 K97C Acrylodan 5-IAF 519 465 6.15 6.16 Ada15.0 Q99C Acrylodan 5-IAF 523 540 6.54 6.83 Ada16.0 A106C Acrylodan 5-IAF 523 465 6.26 6.32 Ada3.0 D11C Pacific Blue 5-IAF 455 520 5.68 6.0 Ada4.0 T15C Pacific Blue 5-IAF 455 523 5.85 6.21 Ada5.0 D21C Pacific Blue 5-IAF 455 523 5.88 6.23 Ada6.0 W32C Pacific Blue 5-IAF 455 520 5.92 6.18 Ada7.0 M38C Pacific Blue 5-IAF 455 520 6.0 6.21 Ada8.0 E45C Pacific Blue 5-IAF 455 520 5.89 6.11 Ada9.0 K53C Pacific Blue 5-IAF 455 520 5.73 6.0 Ada10.0 Q63C Pacific Blue 5-IAF 455 523 5.76 6.27 Ada11.0 Y71C Pacific Blue 5-IAF 455 520 5.83 6.13 Ada12.0 V87C Pacific Blue 5-IAF 455 525 5.78 6.17 Ada13.0 V92C Pacific Blue 5-IAF 455 523 5.87 6.23 Ada14.0 K97C Pacific Blue 5-IAF 455 523 5.83 6.23 Ada15.0 Q99C Pacific Blue 5-IAF 455 523 5.89 6.2 Ada16.0 A106C Pacific Blue 5-IAF 455 523 5.8 6.26 aDetermined by fitting ratiometric signal of the intensities measured at Xi and 2.2 to equations 20-25.
b Poor fit and/or noisy data.
The response of Adaptor2.0 to pH.
The single primary amine at the amino terminus of each lysine-free Adaptor2.0 proteins was labeled with the amine-reactive Fluorescein derivative FAM.
Acrylodan was attached to the disulfide in a second labeling reaction. Adaptor2.0a and 2.0b both exhibited pH-sensitive ngmFRET between the Acrylodan donor and Fluorescein acceptor (FIG. 26A-B). Adaptor2.0a was more soluble than Adaptor2.0b at low pH values, consistent with their difference in number of surface charged groups.
The relatively high Acrylodan donor emission intensity indicates that the ngmFRET
efficiency of Adaptor2.0 is less than that of the Adaptor1.0 constructs. This is likely to be a consequence of orientation effects, as the distances between the donors and acceptors are similar in both adaptor proteins. Furthermore, the donor exhibits two emission peaks, suggesting that the Acrylodan conjugate is located in multiple environments, or adopts both twisted and planar conformations (see above). These results indicate that the environment of the disulfide attachment point differs in these two adaptor proteins, which may be a consequence of the surface redecoration with arginine residues.
The intensity of the shorter wavelength donor peak (460 nm), which is well-separated from the main Fluorescein emission wavelength (520 nm), does not change in response to pH, suggesting that the ngmFRET efficiency remains constant, consistent with a mechanism based on alteration of the non-radiative decay of the Fluorescein in response to pH, as was the case for the Adaptor1.0 conjugates. The intensities of neither the acceptor emission, nor its 500 nm absorption band exhibit large dependencies on pH, although the direction of change is the same as for Adpator1Ø This suggests that the effective charge on the phenolate is reduced in this construct, consistent with formation a hydrogen bond or salt bridge. K36 is located near to the ecTRX N-terminus (FIG. 22), and is a possibly candidate for forming such an interaction (K57 is adjacent to the N-terminus, but has been mutated to methionine in the adaptor proteins).
Adaptor protein thermostability.
The temperature dependence of the pH responses was determined for the Adaptor1.0 R73C=Pacific Blue, disulfide=Fluorescein conjugate using a Roche LightCycler (FIG. 27).
The thermostability of the conjugate is high, increasing from ¨87 Cat pH 4.5 to >100 C at neutral pH and above. The temperature dependence of the pKa value is modest, with a ACp =
0.09 kcal/mol/K.
The thermostability of the Adaptor2.0=Fluorecein, disulfide=Acrylodan is ¨60 Cat neutral pH (FIG. 27). Unlike Adaptor1.0, thermostability does not vary with pH. The temperature dependence of the pKa also is modest, with a ACp = 0.004 kcal/mol/K.
The effect of the adaptor protein on Fluorescein pKa values.
The pKa values of the excited states, as determined from the emission spectra, are consistently lower than the corresponding ground state values obtained from the absorption spectra (Table 9). This effect is consistent with a Forster cycle in which the excited state base has a longer emission wavelength than its conjugate acid such that excited state is more acidic than the ground state (Valeur 2012).
The excited and ground state pKa values of the Adaptor2.0 conjugates are significantly lower (-0.4 pH units) than those of Adaptor1.0 (Table 9). These results indicate that the phenolate is stabilized by the Adaptor2.0 protein matrix, consistent with a proposed direct interaction with a protein side-chain (see above).
EXAMPLE 4. MATERIALS AND METHODS FOR EXAMPLES 1-3 Gene construction. In all constructs used in this study open reading frames (ORFs) encoding engineered proteins were placed behind an efficient Shine-Dalgamo ribosome-binding site, and flanked by a T7 promoter and terminator at the 5' and 3' ends respectively, using the GeneFab program(Cox et al. 2007). All DNA sequences were first designed in silico, and then synthesized by oligonucleotide assembly and cloned into pUC57 by GeneWiz, Inc. (South Plainfield, New Jersey).
The E. coli thioredoxin gene (ecTRX) DNA sequence was taken from the electronic genomic sequence NC_012947, protein identifier YP_003038425.1. Publicly available genome sequences were obtained from the National Center of Biotechnology Information (ftp://ftvp.ncbi.nih.gov/genomes/Bacteria/all.gbk.tar.gz). The T.
thermosaccharolyticum glucose-galactose-binding protein, ttGGBP amino acid sequence was taken from genome NC _ 014410, protein identifier YP_ 003852930.1. The putative leader peptide that mediates secrection of ttGGBP into the periplasm was identified by alignment with the mature form of the Escherichia coli glucose-galactose binding protein, ecGGBP, and removing the 49 amino acids N-terminal to the start of the aligned mature ecGGBP amino acid sequence. A single endogenous cysteine residue (C207 in the mature protein) was mutated to alanine, so that further cysteine mutants would install a unique thiol for site-specific labeling with fluorophores. The 18-residue PZif amino acid sequence is or comprises TGEKPYKCPECGKSFSRS (SEQ ID NO: 42) (Smith et al. 2005). The Yellow Fluorescent Protein amino acid sequence is described in (SEQ ID NO: 149) (Grimley et al.
2013).
The wild-type genomic ecTRX DNA sequence was used to designs the adaptor mutants and the fusion proteins described in this study. The ttGGBP or YFP
amino acid sequences were back-translated into DNA optimized in context of the expression construct by the OrfOpt or program designed to predict highly expressed mRNA sequences in E. coli. The OrfOpt simultaneously imposes AU-rich nucleotide composition at the 5' and 3' ends of the ORF, low RNA secondary structure content and favorable codon usage (Allert et al. 2010).
Subsequent single and multiple point mutations were designed by preparing mutant sequences of the synthetic ORF' sequences using GfMutagenesis, an in-house program that introduces point mutations into an ORF' using the most prevalent codon in E.
coli for an amino acid. To design the sequences of the ttGGBP fusions with the PZif peptide, the peptide sequence was first placed behind the optimized ttGGBP sequence; the DNA sequence of the resulting fusion protein was then re-optimized with OrfOpt without changing the optimized ttGGBP portion. The N- and C-terminal fusions of ecTRX and ttGGBP
mutants were designed by joining the respective wild-type and optimized DNA sequences without further manipulation. All designed protein sequences were terminated with a C-terminal affinity purification tag comprising hexa-histidine peptide placed behind a GGS linker to enable metal-mediated affinity purification(Hengen 1995).
Protein expression, purification, and fluorescent conjugate preparation.
Proteins were prepared by fermentation in Escherichia coli and purified by immobilized metal affinity chromatography (IMAC) as described(Sameiro 2009). In the final purification step, unlabeled proteins are immobilized on the IMAC beads and labeled overnight (4 C, rotating end-over-end) with a thiol-reactive fluorophore (five-fold stoichiometric excess over protein).
Following two rinses with buffer (100 mM NaC1, 1mM CaC12, 20 mM MOPS, pH 6.9) to remove unincorporated label, the proteins were either labeled with a second fluorophore, or eluted from the beads. In experiments that required a second labeling step, the immobilized protein was first extensively washed (5x10 mL), followed by reduction of the cysteine residues in the PZif disulfide-containing by addition of 1 mM TCEP. After one hour, the protein was washed (5x10 mL), the second fluorophore was added, and incubated as described above. To elute labeled protein from the IMAC beads, 6 mL of elution buffer (400 mM imidazole, 500 mM NaC1, 1mM CaC12, 20 mM MOPS, pH 7.8) was added, and the beads removed by centrifugation. Following dialysis of the eluate against three changes of assay buffer (20 mM KC1, 1 mM CaC12, 20 mM MOPS, pH 7.4), using 10 kDa semi-perimeable membrane (Snake Skin), the fluorescent conjugates were concentrated in a 10 kDa cutoff spin concentrator (Vivaspin, GE Healthcare). Their purity was assessed by SDS/PAGE.
Preparation of titration series to measure ligand-binding. 12-, 24-, or 48-point logarithmic titration series were prepared on a Tecan Freedom liquid-handling robot, using an in-house program, `TitrationPlate', that compiles an abstract description of a multi-component titration series into machine instructions for operating the robot.
For glucose titrations in ttGGBP constructs, concentrations were varied from 0-1.7 M in 20 mM KC1, 20 mM MOPS (pH 7.4) supplemented with either 1 mM EGTA or 1 mM CaC12. For chloride titrations in C1BP constructs, concentrations were varied from 0-1.95 M in 35 mM potassium gluconate, 20 mM MOPS (pH 7.4). For pH titrations in adaptor constructs, pH
was varied from 4.0-9.5 in 20 mM KC1 and 20 mM MOPS.
Determination of emission intensity spectra. Ligand- and wavelength-dependent emission intensities were recorded on a Nanodrop3300 (Thermo Scientific) at room temperature. Using the LED closest to the optimal excitation wavelength of the fluorophore (UV, 365 nm; blue, 470 nm; 'white', 460-550 nm).
Ratiometric analysis of ligand binding. Isothermal urea titrations were extracted from the fluorescent landscape or emission spectra datasets obtained as described above.
Monochromatic emission intensities Ix (these intensities correspond to a bandpass intensity, recorded either with a physical, or by integrating in the interval X-8, k-F8 in the case of an emission spectrum), were fit to I2=aP'9)62(1¨ Y trujEsaffi A5, frue 12 where ap0/32 and s are the fluorescence baselines associated with the ligand-free and ligand-bound states of the protein, respectively, and yfrue the fractional saturation of the protein(Layton and Hellinga 2010). Baseline functions can be constant, linear, or a second-order polynomial. For the ligand- and temperature-dependent fluorescence landscapes, we use a constant value for aP )51 x , but sat 16 x is described by a linear dependence on urea concentration, [L]:
sat fix =ax bx[L] 21 For a single ligand-binding site, the fractional saturation is given by [L]
Y =r , 22 Lid+ K d where [L] is the ligand concentration and K d the dissociation constant, "elCd for frue.
A ratiometric signal at a given point in a titration series, R12(t), is given by the ratio of intensities at two wavelengths, 67(21,t), 67(22,0 in the emission spectrum measured at that point:
õ obs rt iii,t) R12(t), "t A' 23 , obs r( a f '"t '\"29`) where a t is an attenuation factor that describes the effect of variations in sample size (i.e. the amount of observable fluorophore) in the tth sample on the wavelength-independent intensity of the entire emission spectrum. This signal removes wavelength-independent emission intensity attenuation effects due to variations in conjugate concentration, photobleaching, fluctuations in excitation source intensities, and detection efficiency(Demchenko 2010, Demchenko 2014). It is a key aspect for high-precision sensing using the reagentless fluorescently-responsive sensors described here. The ratiometric signal also can be fit to a binding isotherm:
R1,2 =aP/a R(1¨ YR)+satfiRY R 24 where aP fiR and sat/3R are the baselines, and R the apparent fractional saturation of the protein (with aPPKd ). In general, irueKd #aPPKd ; if both baselines are constant, a simple relationship can be derived relating aPPKd to "e1Cd(Grim1ey et al. 2013):
P I
a n WK. d d sat i="elµ n 25 where aP A2 and sat/22 are the emission intensities of the monochromatic signal at wavelength X2 of the ligand-free and ligand-bound protein, respectively.
Following a fit of the titration series using equations 20-24, at values can be recovered by taking the average comparison of the observed and calculated intensities at the two wavelengths:
1 ( cak Aili,t) cak 10,2,1 a = ___________ 1 + __ n 26 t 2 obsi(i,t) 0õ702,t) The at value can then be applied to all wavelengths to obtain an emission spectrum or integrated intensity of the tth titration point corrected for variations in sample size:
corr i(A)= at obs/(2) 27 where "r7(2) and bs/(2) are the wavelength-dependent intensities of the corrected and observed emission spectra, respectively.
The fractional error in the chemometric concentration measurement, depends on the first derivative of the binding isotherm as follows(Marvin et al. 1997):
A
as ei 2 =-xí'1,2 ) _______________________________________________________________ ¨ = = X

S S dS ) Where R1,2 is the ratiometric signal (equation 23), ei,2 its experimental error, and ciS is the resulting chemometric error in the concentration. We can then define a relative precision function P
where P(S) is the relative precision at concentration S, which reaches a maximum value (i.e.
lowest error), Pmax, at the Ka.
For a given isothermal titration, values for aPPKd and "elCd were obtained using a non-linear fitting algorithm in which these two parameters were simultaneously fit to the three experimental binding isotherms using equations 20 and 24, with the two monochromatic isotherms sharing the same "elCd value. Three separate pairs of aP 16 and "tfl were fit in this procedure, corresponding to the two monochromatic and the ratiometic signals, respectively.
Two distinct ratiometric response models can be used: coupled (both wavelengths respond to ligand); uncoupled ( the second wavelength is non-responsive; i.e. remains constant).
Optionally, an attenuation vector, a(t) containing at values for each titration point (equation 26), can be refined by iterative fit cycles in which the a(t) vector of a previous cycle is used to adjust the integrated intensities of the next cycle. The program `Nanodrop3300' was used analyze ligand binding.
Analysis of emission spectra components. Wavelength-dependent,/( k),emission intensities at were converted to wavenumber-dependent intensities(Valeur 2012, Lakowicz 2006), /(v):
/(v) = 22/(2) 30 Singular value decomposition was used for model-free identification of regions in the emission spectra that vary with respect to glucose concentration(Henry 1992).
An A. data matrix was constructed by recording /(v) values of m frequencies in columns for n titration points in rows. This matrix was decomposed as A. =U .S nnVn: 31 where Unin records n spectral components at m frequencies ranked by the weight of their contribution to the reconstruction of the experimental data, Vnn records the contribution of the nth component to the nth titration point, Snn records the weight of the nth component.
Decomposition was carried using the in-house Nanodrop3300 program, written in Python.
The linalg.svd method in the open-source Python scipy package (www.scipy.org, version 0.7.2) was used to solve the decomposition. The relative weight of the nth component in U., fn, was calculated from Snn, by normalizing the values in S with its trace:
fn = Snn(n n) tr(Snn) The fractional states of n individual electronic transitions in a spectrum were determined by fitting n Gaussians(Valeur 2012, Lakowicz 2006) to the emission intensities of the corrected spectra (equation 27) transformed into the frequency domain (equation 30):
Cakii (V)_ E
i=n Ai e 2( 0 - 33 1 -Nlr where pi is the wavenumber corresponding to the peak intensity of the ith transition, Ai the area contributed to the total spectrum by this transition, and a-the spectral width of all transitions. The fraction, f;, of the ith transition is given by:
Ai A =
i=n 34 EA;
j=1 Wavelength dependent residuals are given by:
6.6/V's/H¨cak/(v) 35 Fits were carried out by minimizing the least squares difference between observed and calculated spectra, using simplex and conjugate gradient methods implemented in Nanodrop3300 (scipy package methods optimizelmin and optimizeleastsq, respectively).
For titration series with N spectra, collected as a function of titrant concentration, global fits were used in which, as a first approximation, gi values were kept identical in the apo-protein and saturated glucose complex, and a-was universal for all transitions in all spectra. Au values were allowed to vary in each kth spectrum. The variation of the fraction for each transition,f,k, was then fit to a binding isotherm (equation 20), constraining the fit aPPKd value to be common to all transitions. These analyses revealed the shifts in transition dipole populations in response to ligand binding.
Analysis of ligand-binding properties using thermal melts. Protein thermal stabilities were determined by measuring the temperature-dependence of the fluorescence signal, using a Roche LightCycler (Layton and Hellinga 2010). The total fluorescence intensity, S, is given by S = )0F fF fiu fu 36 wherefF and fu are the fractions of protein in the folded and unfolded states, respectively, and F and flu the fluorescence baselines of these two states. To get the fractions of the two states, we have fN = 1+ Ku(T) and fu =1- fN 37 where Ku(T) is the temperature-dependent unfolding equilibrium constant, which by the van't Hoff approximation is given by )IR
Ku = e TTm38 Where T is the temperature, T,õ the unfolding reaction transition mid-point temperature, and AHu the enthalpy of unfolding.
To obtain the temperature dependence of the binding reaction, the Kd values of all the individually determined isotherms were fit the Gibbs-Hemholtz equation(Layton and Hellinga 2010):
AG: (T) =A ref1-1: + ACp,b ¨ Tr4. )¨ T AS + AC ln 39 p,b 7, A rff where AG: (T) is the standard free energy of binding at 1 M ligand at temperature T, AG: (T) = ¨RT141 + _____________________________________________________ 40 d k7' H: and Are f S: the molar enthalpy and entropy of binding, respectively, at the reference temperature, Tref, and ACp,b the heat capacity of the binding reaction. This data analysis was carried out using `TitrationMeltAnalysis'.
RATIOMETRIC ngmFRET BIOSENSORS AND USES THEREOF
The energy transfer between a donor-acceptor pair can be responsive to ligand-mediated changes in the photophysics of a single partner, without involving a change in the geometry (distance, angle) between the two partners (non-geometrically modulated FRET, ngmFRET). Changes in the individual fluorophore properties include alterations in relative rates of excited state radiative and non-radiative decay rates (quenching) and switching between different excited state dipoles (dipole switching). The latter can alter both the emission intensity spectrum, and the absorption spectrum if the ground state electronic structure is affected by ligand binding. The analysis that we present (equations 8-19) shows that the detailed mechanism by which quenching and dipole switching affects ngmFRET
depends on whether the ligand-responsive fluorophore functions as the donor or acceptor in the system (Table 1). Dipole switching affects ngmFRET by altering the spectral overlap between the two partners. Accordingly, if the directly responsive partner is the donor, then effects are seen only if ligand binding modulates the excited state (emission), whereas if it is the acceptor, only ground state (absorption) effects are observable. Quenching (change in the ratio of radiative to non-radiative emission rates) effects in directly responsive donors alter the ngmFRET transfer rate, thereby affecting the emission intensities of both donor and acceptor; directly responsive acceptor quenching affects only its own intensity. The possible choices in quenching, dipole switching and directly responsive partner functional role together combine into sixteen different scenarios by which ngmFRET occurs (Table 1).
We have constructed over 15 different donor-acceptor pairs in three different protein-based, semi-synthetic, fluorescent biosensors, all of which show ratiometric responses to binding of their cognate ligands (Table 11). Together these conjugates represent five of the sixteen possible ngmFRET scenarios. Pure geometry-based (tgmFRET) effects alter the donor and acceptor emission intensities in opposing directions only. Seven conjugates exhibit parallel ligand-mediated intensity increases or decreases, which clearly indicate the presence of non-geometrical ngmFRET effects. Quantitative differential differences between the donor and acceptor changes the shape of the emission spectrum. In three rigid conjugates, the intensity of a donor changes when partnered with directly responsive acceptor. This effect can occur only if the acceptor ground state absorption spectrum changes in response to ligand binding, providing clear evidence for dipole switching effects.

Table 11. Summary of observed intensity response patterns and ngmFRET
mechanisms.
Response pattern' Ligand Donor Acceptor A/D
Construct ratio Modelb YFP 17C.Pacific Bluee Chloride 1' si, si, a+
ttGGBP 11C.Badan gif.5-1AF Glucose 1' si, si, d 0"
ttGGBP 11C.Badan gif. Alexa532 1' si, si, ct ttGGBP 17C.Pacific Blue gif.5-1AF 1' 1' 1' d 0 ttGGBP 17C.Badan f3Zif.5-IAFd 1' 4, 4, ct ttGGBP 17C.Badan gifA1exa532 d 1' 4, 4, dgY
ttGGBP 17C.Badan C- 1' 4, 4, d 0"
ecTRX.Alexa532 ttGGBP 111C.Acrylodan 1' 1' si, d 0 f3Zif. Alexa532 ttGGBP 151C.Acrylodan si, si, 1' d+0 f3Zif. Alexa532 ttGGBP 151C =Badan f3Zif. Alexa532 1' 1' 1' ctO
ttGGBP 182C.Acrylodan gif.5-1AF si, 1' 1' 6'0+
ttGGBP 182C.Acrylodan si, 1' 1' 6'0+
f3Zif. Alexa532d ttGGBP 182C.IAEDANS f3Zif.5-IAFd 1' 1' 4, d4 Adaptor 1.0e disulfide.5-IAF 11+ 1' si, si, a+ 0"
C73 Pacific Blue Adaptor 1.0e disulfide.5-IAF 1' si, si, a+
C73.Acrylodan 'Change in magnitude ( 1', increase; sL, decrease; 0, no change) of the donor and acceptor fluorescence intensities and their ratio.
bSee Table 1 eResponse pattern and model are the same for all members of the chloride-binding protein series C1BP2-14.
dPhase I of the response to glucose (see main text).
eResponse pattern and model are the same for all members of the Adaptor family.
Fluorescently responsive sensors take advantage of ligand-mediated changes in the photophysics of a fluorophore. Analyte binding shifts the population distribution of the two fluorophore states whose photophysical properties are associated with the ligand-free and ¨
bound forms of the sensor, respectively. Ratiometic measurements of differential changes at two distinct wavelengths are optimal for taking advantage of ligand-mediated changes in emission intensities, because these remove unwanted fluctuations in absolute intensity.
Ratiometry is possible only if the shape of the fluorescence emission intensity spectrum differs in the ligand-free and ¨bound states (dichromatic changes). Many sensors are based on fluorescent protein conjugates or chemoresponsive fluorophores.
Unfortunately, the emission intensity spectra of many of these materials change only in intensity and not shape (monochromatic responses), precluding their use in ratiometry. The ngmFRET
systems presented here can readily convert monochromatic into dichromatic responses, thereby overcoming the limitations and drawbacks of earlier systems. Furthermore, we have shown that ratiometric responses in a single-fluorophore conjugate can be improved if the emission of one of the switched dipoles is coupled to a significantly brighter acceptor. We have also shown that optimal ratiometry requires careful tuning of the spectral overlap in an ngmFRET
system such that both donor and acceptor exhibit significant emission intensities.
The ngmFRET approach can be applied to many important sources of fluorescent sensors. Non-limiting examples include ligand binding proteins for sugars (such as glucose, galactose, lactose, arabinose, ribose, and maltose), lactate, urea, anions (e.g., bicarbonate, phosphate, sulfate, and halide anions such as chloride, fluoride, iodide, astatide, ununseptide, and bromide), cations (e.g., calcium and hydrogen ions), dipeptides, amino acids (such as histidine, glutamine, glutamate, aspartate), and elements (e.g., iron).
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OTHER EMBODIMENTS
While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference.
All published foreign patents and patent applications cited herein are hereby incorporated by reference.
Genbank and NCBI submissions indicated by accession number cited herein are hereby incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are hereby incorporated by reference.
While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims

What is claimed is:
1. A method of detecting a ligand in a sample, comprising (a) contacting a biosensor with the ligand, wherein the biosensor comprises a ligand-binding protein, a directly responsive fluorophore and an indirectly responsive fluorophore, the directly responsive and the indirectly responsive fluorophores being located at two distinct sites of the ligand-binding-protein, wherein (i) the directly responsive fluorophore is a donor fluorophore and the indirectly responsive fluorophore is an acceptor fluorophore; or (ii) the directly responsive fluorophore is an acceptor fluorophore and the indirectly responsive fluorophore is a donor fluorophore, (b) contacting the biosensor with radiation comprising a wavelength within the excitation spectrum of the donor fluorophore, wherein (i) a fluorescence property of the directly responsive fluorophore changes in response to ligand binding in the absence or presence of the indirectly responsive fluorophore;
(ii) a fluorescence property of the indirectly responsive fluorophore does not change in response to ligand binding in the absence of the directly responsive fluorophore;
(iii)non-geometrically modulated Förster resonance energy transfer (ngmFRET) occurs between the directly responsive fluorophore and the indirectly responsive fluorophore;
(iv)fluorescent light is emitted from the biosensor, wherein the light emitted from the biosensor comprises a combination of light emitted from the directly responsive fluorophore and light emitted from the indirectly responsive fluorophore; and (v) wherein the ratio of the fluorescence emission intensity emitted from the biosensor at each of two distinct wavelengths changes in response to ligand binding;
(c) measuring fluorescent light that is emitted from the directly responsive fluorophore and the indirectly responsive fluorophore; and (d) calculating a ratiometric signal, thereby detecting the ligand in the sample.

2. The method of claim 1, the ratiometric signal (R1,2) comprises a quotient of two intensities, I.lambda.1 and I.lambda.2, measured at two independent wavelengths, .lambda.1 and .lambda.2 and is calculated according to the following equation:
R1,2 = I.lambda.1 / I.lambda.2 3. The method of claim 1, wherein the directly responsive fluorophore is a donor fluorophore and the indirectly responsive fluorophore is an acceptor fluorophore, and wherein ngmFRET occurs between the donor fluorophore and the acceptor fluorophore when the donor fluorophore is contacted with radiation within its excitation spectrum.
4. The method of claim 1, wherein the directly responsive fluorophore is an acceptor fluorophore and the indirectly responsive fluorophore is a donor fluorophore, and wherein ngmFRET occurs between the donor fluorophore and the acceptor fluorophore when the donor fluorophore is contacted with radiation within its excitation spectrum.
5. The method of claim 1, wherein the change in the fluorescent property of the directly responsive fluorophore comprises (i) a bathochromic or hypsochromic shift in the emission or excitation spectrum thereof; or (ii) a change in the ratio of radiative to non-radiative emission rates thereof.
6. The method of claim 1, wherein (a) the directly responsive fluorophore is Badan and emission intensity is measured at a wavelength of 467 nm, and wherein the indirectly responsive fluorophore is 5-iodoacetamidofluorescein (5-IAF) and emission intensity is measured at a wavelength of 520 nm;
(b) the directly responsive fluorophore is Badan and emission intensity is measured at a wavelength of 467 nm, and wherein the indirectly responsive fluorophore is A1exa532 and emission intensity is measured at a wavelength of 560 nm;
(c) the directly responsive fluorophore is Pacific Blue and emission intensity is measured at a wavelength of 456 nm, and wherein the indirectly responsive fluorophore is 5-IAF and emission intensity is measured at a wavelength of 520 nm;

(d) the directly responsive fluorophore is Acrylodan and emission intensity is measured at a wavelength of 465 nm, and wherein the indirectly responsive fluorophore is 5-IAF and emission intensity is measured at a wavelength of 520 nm;
(e) the directly responsive fluorophore is Acrylodan and emission intensity is measured at a wavelength of 480 nm, and wherein the indirectly responsive fluorophore is Alexa532 and emission intensity is measured at a wavelength of 549 nm;
(f) the directly responsive fluorophore is 5-IAF and emission intensity is measured at a wavelength of 455 nm, and wherein the indirectly responsive fluorophore is Pacific Blue and emission intensity is measured at a wavelength of 520 nm;
(g) the directly responsive fluorophore is Oregon Green and emission intensity is measured at a wavelength of 455 nm, and wherein the indirectly responsive fluorophore is Pacific Blue and emission intensity is measured at a wavelength of 520 nm; or (h) the directly responsive fluorophore is N-(Iodoacetaminoethyl)-1-naphthylamine-5-sulfonic acid (IAEDANS) and emission intensity is measured at a wavelength of 465 nm, and wherein the indirectly responsive fluorophore is 5-IAF and emission intensity is measured at a wavelength of 520 nm.
7. The method of claim 1, wherein (a) the directly responsive fluorophore is a yellow fluorescent protein and emission intensity is measured at a wavelength of 530 nm, and wherein the indirectly responsive fluorophore is Acrylodan and emission intensity is measured at a wavelength of 500 nm; or (b) the directly responsive fluorophore is a yellow fluorescent protein and emission intensity is measured at a wavelength of 530 nm, and wherein the indirectly responsive fluorophore is Pacific Blue and emission intensity is measured at a wavelength of 455 nm.
8. The method of claim 1, wherein the directly responsive fluorophore comprises a donor fluorophore and the indirectly responsive fluorophore comprises an acceptor fluorophore.
9. The method of claim 8, wherein (a) the emission intensity of the donor fluorophore decreases and the emission intensity of the acceptor fluorophore increases upon ligand binding to the ligand-binding protein when the donor fluorophore is contacted with radiation within the excitation spectrum of the donor fluorophore;
(b) the emission intensity of the donor fluorophore increases and the emission intensity of the acceptor fluorophore decreases upon ligand binding to the ligand-binding protein when the donor fluorophore is contacted with radiation within the excitation spectrum of the donor fluorophore;
(c) the emission intensities of the donor fluorophore and the acceptor fluorophore both decrease upon ligand binding to the ligand-binding protein when the donor fluorophore is contacted with radiation within the excitation spectrum of the donor fluorophore;
(d) the emission intensity of the donor fluorophore decreases and the emission intensity of the acceptor fluorophore increases, decreases, or remains about the same upon ligand binding to the ligand-binding protein when the donor fluorophore is contacted with radiation within the excitation spectrum of the donor fluorophore;
(e) the emission intensity of the donor fluorophore increases, decreases, or remains about the same and the emission intensity of the acceptor fluorophore decreases upon ligand binding to the ligand-binding protein when the donor fluorophore is contacted with radiation within the excitation spectrum of the donor fluorophore;
(f) the emission intensities of the donor fluorophore and the acceptor fluorophore both increase upon ligand binding to the ligand-binding protein when the donor fluorophore is contacted with radiation within the excitation spectrum of the donor fluorophore;
(g) the emission intensity of the donor fluorophore increases, decreases, or remains about the same and the emission intensity of the acceptor fluorophore increases upon ligand binding to the ligand-binding protein when the donor fluorophore is contacted with radiation within the excitation spectrum of the donor fluorophore; or (h) the emission intensity of the donor fluorophore increases and the emission intensity of the acceptor fluorophore increases, decreases, or remains about the same upon ligand binding to the ligand-binding protein when the donor fluorophore is contacted with radiation within the excitation spectrum of the donor fluorophore.

10. The method of claim 1, wherein the directly responsive fluorophore comprises an acceptor fluorophore and the indirectly responsive fluorophore comprises a donor fluorophore.
11. The method of claim 10, wherein (a) the emission intensity of the donor fluorophore decreases and the emission intensity of the acceptor fluorophore increases, decreases, or remains about the same upon ligand binding to the ligand-binding protein when the donor fluorophore is contacted with radiation within the excitation spectrum of the donor fluorophore;
(b) the emission intensity of the donor fluorophore increases and the emission intensity of the acceptor fluorophore increases, decreases, or remains about the same upon ligand binding to the ligand-binding protein when the donor fluorophore is contacted with radiation within the excitation spectrum of the donor fluorophore;
(c) the emission intensity of the donor fluorophore remains about the same and the emission intensity of the acceptor fluorophore decreases upon ligand binding to the ligand-binding protein when the donor fluorophore is contacted with radiation within the excitation spectrum of the donor fluorophore;
(d) the emission intensity of the donor fluorophore decreases and the emission intensity of the acceptor fluorophore increases, decreases, or remains about the same upon ligand binding to the ligand-binding protein when the donor fluorophore is contacted with radiation within the excitation spectrum of the donor fluorophore;
(e) the emission intensity of the donor fluorophore increases and the emission intensity of the acceptor fluorophore increases, decreases, or remains about the same upon ligand binding to the ligand-binding protein when the donor fluorophore is contacted with radiation within the excitation spectrum of the donor fluorophore;
(f) the emission intensity of the donor fluorophore remains about the same and the emission intensity of the acceptor fluorophore increases upon ligand binding to the ligand-binding protein when the donor fluorophore is contacted with radiation within the excitation spectrum of the donor fluorophore;
(g) the emission intensity of the donor fluorophore decreases and the emission intensity of the acceptor fluorophore increases upon ligand binding to the ligand-binding protein when the donor fluorophore is contacted with radiation within the excitation spectrum of the donor fluorophore; or (h) the emission intensity of the donor fluorophore increases and the emission intensity of the acceptor fluorophore remains about the same, increases, or decreases upon ligand binding to the ligand-binding protein when the donor fluorophore is contacted with radiation within the excitation spectrum of the donor fluorophore.
12. The method of claim 1, wherein the ligand-binding protein comprises the directly responsive fluorophore.
13. The method of claim 12, wherein the directly responsive fluorophore is formed by an autocatalytic cyclization of an oligopeptide within the ligand-binding protein.
14. The method of claim 13 wherein the oligopeptide is located within an interior a helix.
15. The method of claim 13, wherein the oligopeptide comprises three consecutive residues, four consecutive residues, or five consecutive residues.
16. The method of claim 12, wherein the directly responsive fluorophore is formed by an autocatalytic cyclization of a tripeptide located in an interior a helix of the ligand-binding protein.
17. The method of claim 12, wherein the ligand-binding protein comprises a yellow fluorescent protein (YFP).
18. The method of claim 1, wherein ligand binding causes a change in signaling by the directly responsive fluorophore.
19. The method of claim 14, wherein the indirectly responsive fluorophore is attached to the ligand-binding protein via a covalent bond.
20. The method of claim 19, wherein the covalent bond comprises a disulfide bond, a thioester bond, a thioether bond, an ester bond, an amide bond, or a bond that has been formed by a click reaction.

21. The method of claim 19, wherein the indirectly responsive fluorophore is attached to a cysteine or a lysine of the protein.
22. The method of claim 19, wherein the indirectly responsive fluorophore is attached to the N-terminus or the C-terminus of the protein.
23. The method of claim 19, wherein the indirectly responsive fluorophore is attached to the N-terminus or the C-terminus of the protein via a fluorophore attachment motif.
24. The method of claim 23, wherein the fluorophore attachment motif comprises an amino acid or a polypeptide.
25. The method of claim 24, wherein the polypeptide comprises amino acids in the sequence of .beta.Zif (SEQ lD NO: 42).
26. The method of claim 24, wherein the polypeptide comprises a stretch of at least 50, 60, 70, 80, 90, or 100 amino acids in a sequence that is at least about 85%, 90%, 95%, or 99% identical to the amino acid sequence of E. coli thioredoxin (ecTRX; SEQ lD
NO: 151).
27. A method of detecting a ligand in a sample, comprising (a) contacting a biosensor with the ligand, wherein the biosensor comprises an amino acid or a polypeptide, a directly responsive fluorophore and an indirectly responsive fluorophore, the directly responsive and the indirectly responsive fluorophores being located at two distinct sites of the amino acid or polypeptide, wherein the directly responsive fluorophore is chemoresponsive, and wherein (i) the directly responsive fluorophore is a donor fluorophore and the indirectly responsive fluorophore is an acceptor fluorophore; or (ii) the directly responsive fluorophore is an acceptor fluorophore and the indirectly responsive fluorophore is a donor fluorophore, (b) contacting the biosensor with radiation comprising a wavelength within the excitation spectrum of the donor fluorophore, wherein (i) a fluorescence property of the directly responsive fluorophore changes in response to ligand binding in the absence or presence of the indirectly responsive fluorophore;
(ii) a fluorescence property of the indirectly responsive fluorophore does not change in response to ligand binding in the absence of the directly responsive fluorophore;
(iii)ngmFRET occurs between the directly responsive fluorophore and the indirectly responsive fluorophore;
(iv)fluorescent light is emitted from the biosensor, wherein the light emitted from the biosensor comprises a combination of light emitted from the directly responsive fluorophore and light emitted from the indirectly responsive fluorophore; and (v) wherein the ratio of the fluorescence emission intensity emitted from the biosensor at each of two distinct wavelengths changes in response to ligand binding;
(c) measuring fluorescent light that is emitted from the directly responsive fluorophore and the indirectly responsive fluorophore; and (d) calculating a ratiometric signal, thereby detecting the ligand in the sample.
28. The method of claim 27, the ratiometric signal (R1,2) comprises a quotient of two intensities, I.lambda.1 and I.lambda.2, measured at two independent wavelengths, .lambda.1 and .lambda.2 and is calculated according to the following equation:
R1,2 = I.lambda.1/I.lambda.2 .
29. The method of claim 27, wherein the directly responsive fluorophore is a donor fluorophore and the indirectly responsive fluorophore is an acceptor fluorophore, and wherein ngmFRET occurs between the donor fluorophore and the acceptor fluorophore when the donor fluorophore is contacted with radiation within its excitation spectrum.
30. The method of claim 27, wherein the directly responsive fluorophore is an acceptor fluorophore and the indirectly responsive fluorophore is a donor fluorophore, and wherein ngmFRET occurs between the donor fluorophore and the acceptor fluorophore when the donor fluorophore is contacted with radiation within its excitation spectrum.
31. The method of claim 27, wherein the change in the fluorescent property of the directly responsive fluorophore comprises (i) a bathochromic or hypsochromic shift in the emission or excitation spectrum thereof; or (ii) a change in the ratio of radiative to non-radiative emission rates thereof.
32. The method of claim 27, wherein (a) the directly responsive fluorophore is 5-IAF and emission intensity is measured at a wavelength of 460 nm, and wherein the indirectly responsive fluorophore is Acrylodan and emission intensity is measured at a wavelength of 523 nm; or (b) the directly responsive fluorophore is 5-IAF and emission intensity is measured at a wavelength of 520 nm, and wherein the indirectly responsive fluorophore is Pacific Blue and emission intensity is measured at a wavelength of 455 nm.
33. The method of claim 27, wherein the amino acid or the polypeptide comprises 1 amino acid, or a stretch of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 500, 750, or 1000 amino acids.
34. The method of claim 33, wherein the polypeptide comprises a stretch of at least 50, 60, 70, 80, 90, or 100 amino acids in a sequence that is at least about 85%, 90%, 95%, or 99% identical to the amino acid sequence of ecTRX (SEQ lD NO: 151).
35. The method of claim 33, wherein the amino acid or the polypeptide comprises (a) at least 1, 2, or 3 thiol groups;
(b) at least 1, 2, or 3 cysteines that each comprise a sulfhydryl group;
(c) at least 1, 2, or 3 primary amine groups; or (d) at least 1, 2, or 3 lysines that each comprise a primary amine.
36. The method of claim 34, wherein there is no disulfide bond between cysteines within the amino acid sequence of the polypeptide.

37. The method of claim 34, wherein the polypeptide comprises a mutant of ecTRX
comprising a D3X, K4X, K19X, D27X, K37X, K53X, K58X, K70X, R74X, K83X, K91X, K97X, or K101X mutation, or any combination thereof, wherein X is any amino acid, and wherein each ecTRX amino acid position is numbered as in SEQ ID NO: 151.
38. The method of claim 37, wherein the polypeptide comprises a mutant of ecTRX
comprising a D3A, K4R, K4Q, K19R, K19Q, D27A, K37R, K53M, K53R, K58M, K7OR, R74C, K83R, K91R, K97R, or K101R mutation, or any combination thereof, wherein each ecTRX amino acid position is numbered as in SEQ ID NO: 151.
39. The method of claim 34, wherein the polypeptide comprises a mutant of ecTRX that does not comprise a lysine.
40. The method of claim 34, wherein the polypeptide further comprises a hexahistidine tag.
41. The method of claim 34, comprising amino acids in the sequence set forth as any one of SEQ ID NOS: 24-41.
42. The method of claim 27, which wherein the ligand comprises a hydrogen ion.
43. The method of claim 42, which is a biosensor for pH, wherein the directly responsive fluorophore is pH-sensitive.
44. The method of claim 43, wherein the fully excited emission intensity of the directly responsive fluorophore is different at a pH less than about 7.0 compared to a pH of 7.5.
45. The method of claim 43, wherein the directly responsive fluorophore comprises a pH-sensitive fluorophore comprising fluorescein or a derivative thereof.
46. The method of claim 45, wherein the directly responsive fluorophore transitions from a monoanion to a dianion at a pH that is less than 7.0 in an aqueous solution.

47. The method of claim 1, wherein the directly responsive fluorophore is attached to the ligand-binding protein via a covalent bond.
48. The method of claim 47, wherein the covalent bond comprises a disulfide bond, a thioester bond, a thioether bond, an ester bond, an amide bond, or a bond that has been formed by a click reaction.
49. The method of claim 47, wherein the directly responsive fluorophore is attached to a cysteine or a lysine of the protein.
50. The method of claim 47, wherein the indirectly responsive fluorophore is attached to the N-terminus or the C-terminus of the protein.
51. The method of claim 27, wherein the indirectly responsive fluorophore is attached to the N-terminus or the C-terminus of the protein via a fluorophore attachment motif.
52. The method of claim 27, wherein the fluorophore attachment motif comprises an amino acid or a polypeptide.
53. The method of claim 52, wherein the polypeptide comprises amino acids in the sequence of .beta.Zif (SEQ lD NO: 42).
54. The method of claim 52, wherein the polypeptide comprises a stretch of at least 50, 60, 70, 80, 90, or 100 amino acids in a sequence that is at least about 85%, 90%, 95%, or 99% identical to the amino acid sequence of E. coli thioredoxin (ecTRX; SEQ lD
NO: 151).
55. The method of claim 1, wherein if the acceptor fluorophore comprises ruthenium or osmium, then the acceptor fluorophore is not attached to the amino group of the N-terminus of the ligand-binding protein.
56. The method of claim 1, wherein the biosensor does not comprise an E.
coli glutamine-binding protein with Acrylodan attached to 179C.

57. The method of claim 1, wherein the biosensor does not comprise E. coli glucose-binding protein with Aciylodan attached to 255C.
58. The biosensor of claim 1, wherein an overlap of the emission spectrum of the donor fluorophore and the excitation spectrum of the acceptor fluorophore increases upon ligand binding.
59. The method of claim 58, wherein (i) the directly responsive fluorophore comprises the donor fluorophore, and the increase results from a bathochromic shift in the emission spectrum of the donor fluorophore; or (ii) the directly responsive fluorophore comprises the acceptor fluorophore, and the increase results from a hypsochromic shift in the excitation spectrum of the acceptor fluorophore.
60. The method of claim 1, wherein an overlap of the emission spectrum of the donor fluorophore and the excitation spectrum of the acceptor fluorophore decreases upon ligand binding.
61. The method of claim 60, wherein (i) the directly responsive fluorophore comprises the donor fluorophore, and the decrease results from a hypsochromic shift in the emission spectrum of the donor fluorophore; or (ii) the directly responsive fluorophore comprises the acceptor fluorophore, and the decrease results from a bathochromic shift in the excitation spectrum of the acceptor fluorophore.
62. The method of claim 1, wherein the directly responsive fluorophore has a monochromatic spectral change upon ligand binding.
63. The method of claim 1, wherein the directly responsive fluorophore has a dichromatic spectral change upon ligand binding.

64. The method of claim 1, wherein the emission intensity of the donor fluorophore and/or the acceptor fluorophore increases in two phases as ligand concentration increases.
65. The method of claim 1, wherein the ratio of radiative to non-radiative emission or intensity of the directly responsive fluorophore increases by at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 50%, 75%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold upon ligand binding to the ligand-binding protein.
66. The method of claim 1, wherein the ratio of radiative to non-radiative emission or intensity of the directly responsive fluorophore decreases by at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 50%, 75%, 90%, 95%, or 99% upon ligand binding to the ligand-binding protein.
67. The method of claim 1, wherein the directly responsive fluorophore and the indirectly responsive fluorophore are not a naphthalene derivative.
68. The method of claim 1, wherein the directly responsive fluorophore and the indirectly responsive fluorophore are not Prodan, Acrylodan, or Badan.
69. The method of claim 1, wherein the directly responsive fluorophore is not a naphthalene derivative.
70. The method of claim 1, wherein the directly responsive fluorophore is not Prodan, Aciylodan, or Badan.
71. The method of claim 1, wherein the directly responsive fluorophore comprises xanthene, a xanthene derivative, fluorescein, a fluorescein derivative, coumarin, a coumarin derivative, cyanine, a cyanine derivative, rhodamine, a rhodamine derivative, phenoxazine, a phenoxazine derivative, squaraine, a squaraine derivative, coumarin, a coumarin derivative, oxadiazole, an oxadiazole derivative, anthracene, an anthracene derivative, a boradiazaindacine (BODIPY) family fluorophore, pyrene, a pyrene derivative, acridine, an acridine derivative, arylmethine, an arylmethine derivative, tetrapyrrole, or a tetrapyrrole derivative.

72. The method of claim 71, wherein the directly responsive fluorophore comprises fluorescein or a derivative thereof.
73. The method of claim 1, wherein the ligand-binding protein is selected from the group consisting of a glucose-galactose binding protein (GGBP), a glucose-binding protein, a urea-binding protein (UBP), a lactate-binding protein (LacBP), a calcium-binding protein, a calcium-bicarbonate binding protein (BicarbBP), and an iron-bicarbonate binding protein (FeBP).
74. The method of claim 1, wherein (a) the donor fluorophore comprises Pacific Blue and the acceptor fluorophore comprises 5-IAF or 6-IAF;
(b) the donor fluorophore comprises Pacific Blue and the acceptor fluorophore comprises Oregon Green;
(c) the donor fluorophore comprises IAEDANS and the acceptor fluorophore comprises 5-IAF or 6-IAF;
(d) the donor fluorophore comprises acrylodan and the acceptor fluorophore comprises Alexa532;
(e) the donor fluorophore comprises acrylodan and the acceptor fluorophore comprises 5-IAF or 6-IAF;
(f) the donor fluorophore comprises acrylodan and the acceptor fluorophore comprises Pacific Blue or YFP;
(g) the donor fluorophore comprises 5-IAF or 6-IAF and the acceptor fluorophore comprises Pacific Blue;
(h) the donor fluorophore comprises badan and the acceptor fluorophore comprises 5-IAF or 6-IAF; or (i) the donor fluorophore comprises badan and the acceptor fluorophore comprises Alexa532.
75. A biosensor for a ligand comprising a ligand-binding protein, a directly responsive fluorophore and an indirectly responsive fluorophore, the directly responsive and the indirectly responsive fluorophores being located at two distinct sites of the ligand-binding protein, wherein (i) the directly responsive fluorophore is a donor fluorophore and the indirectly responsive fluorophore is an acceptor fluorophore; or (ii) the directly responsive fluorophore is an acceptor fluorophore and the indirectly responsive fluorophore is an donor fluorophore, and wherein if the acceptor fluorophore comprises ruthenium or osmium, then the acceptor fluorophore is not attached to the amino group of the N-terminus of the ligand-binding protein.
76. The biosensor of claim 75, wherein the ligand-binding protein comprises the directly responsive fluorophore.
77. The biosensor of claim 76, wherein the directly responsive fluorophore is formed by an autocatalytic cyclization of an oligopeptide within the ligand-binding protein.
78. The biosensor of claim 76, wherein the ligand-binding protein comprises a Yellow Fluorescent Protein (YFP; SEQ ID NO: 149) or a fluorescent mutant thereof.
79. The biosensor of claim 76, wherein the ligand comprises a halide anion.
80. The biosensor of claim 79, wherein the halide anion comprises a fluoride (F), chloride (CF), a bromide (BC), an iodide (I-), an astatide (AC) anion, or an ununseptide (Ts-) anion.
81. The biosensor of claim 78, wherein the mutant comprises a mutation that alters the interaction of the mutant with a bound halide anion compared to YFP.
82. The biosensor of clam 78, wherein the mutant comprises a mutation that alters the affinity and/or specificity of the mutant for a halide anion compared to YFP.
83. The biosensor of claim 79, comprising 1 halide anion binding site.
84. The biosensor of claim 79, comprising at least 2, 3, 4, or 5 halide anion binding sites.
85. The biosensor of claim 78, wherein at least one amino acid of the YFP
or the fluorescent mutant thereof has been substituted with a cysteine.

86. The biosensor of claim 85, wherein the cysteine is within a first .beta.-strand (.beta.1), a second .beta.-strand (.beta.2) a third .beta.-strand (.beta.3), a fourth .beta.-strand (.beta.4), a fifth .beta.-strand (.beta.5), a sixth .beta.-strand (.beta.6), a seventh .beta.-strand (.beta.), an eighth .beta.-strand (.beta.8), a ninth .beta.-strand (.beta.9), a tenth .beta.-strand (.beta.10), or an eleventh .beta.-strand (.beta.11) of the YFP or the fluorescent mutant thereof.
87. The biosensor of claim 78, comprising one or more of the following substitutions:
E17X, E32X, T43X, F64X, G65X, L68X, Q69X, A72X, H77X, K79X, R80X, E95X, R109X, R122X, D133X, H148X, N149X, V163X, N164X, D173X, Y182X, Q183X, Y203X, Q204X, L221X, and H231X, wherein X is any amino acid, wherein each YFP amino acid position is numbered as in SEQ ID NO: 150.
88. The biosensor of claim 87, wherein X is C.
89. The biosensor of claim 87, comprising one or more of the following substitutions:
F64L, G65T, L68V, Q69T, A725, K79R, R80Q, H148Q, H148G, V163A, H231L, H148Q, or Q183A, wherein each YFP amino acid position is numbered as in SEQ ID NO:
150.
90. The biosensor of claim 87, comprising an R at the 96 position, a Y at the 203 position, a S at the 205 position, and an E at the 222 position, wherein each YFP amino acid position is numbered as in SEQ ID NO: 150.
91. The biosensor of claim 75, wherein ligand binding causes a change in signaling by the directly responsive fluorophore.
92. The biosensor of claim 91, wherein the ligand-binding protein comprises a mutation compared to a naturally occurring protein.
93. The biosensor of claim 92, wherein at least one amino acid of the ligand-binding protein has been substituted with a cysteine.
94. The method of claim 92, wherein the ligand-binding protein comprises a mutant of a microbial ligand-binding protein.

95. The method of claim 94, wherein the ligand-binding protein comprises a mutant of a microbial periplasmic ligand-binding protein.
96. The method of claim 91, wherein the ligand comprises glucose, galactose, lactose, arabinose, ribose, maltose, lactate, urea, bicarbonate, phosphate, sulfate, chloride, fluoride, iodide, astatide, ununseptide, bromide, calcium, a hydrogen ion, a dipeptide, histidine, glutamine, glutamate, aspartate, or iron.
97. The biosensor of claim 96, wherein the ligand-binding protein comprises a GGBP.
98. The biosensor of claim 97, wherein the GGBP is or is a mutant of: an Escherichia sp.
GGBP; a Thermoanaerobacter sp. GGBP; a Clostridium sp. GGBP; a Salmonella sp.
GGBP;
a Caldicellulosiruptor sp. GGBP; a Paenibacillus sp. GGBP; a Butyrivibrio sp.
GGBP; a Roseburia sp. GGBP; a Faecalibacterium sp. GGBP; an Erysipelothrix sp. GGBP;
or an Eubacterium sp. GGBP.
99. A biosensor for a ligand comprising an amino acid or a polypeptide, a directly responsive fluorophore and an indirectly responsive fluorophore, the directly responsive and the indirectly responsive fluorophores being located at two distinct sites of the amino acid or polypeptide, wherein the directly responsive fluorophore is chemoresponsive, and wherein (i) the directly responsive fluorophore is a donor fluorophore and the indirectly responsive fluorophore is an acceptor fluorophore; or (ii) the directly responsive fluorophore is an acceptor fluorophore and the indirectly responsive fluorophore is an donor fluorophore.
100. The method of claim 99, wherein the amino acid or the polypeptide comprises 1 amino acid, or a stretch of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 500, 750, or 1000 amino acids.
101. The biosensor of claim 100, wherein the polypeptide comprises a stretch of at least 50, 60, 70, 80, 90, or 100 amino acids in a sequence that is at least about 85%, 90%, 95%, or 99% identical to the amino acid sequence of ecTRX (SEQ ID NO: 151).

102. The biosensor of claim 100, wherein the amino acid or the polypeptide comprises (a) at least 1, 2, or 3 thiol groups;
(b) at least 1, 2, or 3 cysteines that each comprise a sulfhydryl group;
(c) at least 1, 2, or 3 primary amine groups; or (d) at least 1, 2, or 3 lysines that each comprise a primary amine.
103. The biosensor of claim 100, wherein there is no disulfide bond between cysteines within the amino acid sequence of the polypeptide.
104. The biosensor of claim 101, wherein the polypeptide comprises a mutant of ecTRX
comprising a D3X, K4X, K19X, D27X, K37X, K53X, K58X, K70X, R74X, K83X, K91X, K97X, or K101X mutation, or any combination thereof, wherein X is any amino acid, and wherein each ecTRX amino acid position is numbered as in SEQ lD NO: 151.
105. The biosensor of claim 104, wherein the polypeptide comprises a mutant of ecTRX
comprising a D3A, K4R, K4Q, K19R, K19Q, D27A, K37R, K53M, K53R, K58M, K7OR, R74C, K83R, K91R, K97R, or K101R mutation, or any combination thereof, wherein each ecTRX amino acid position is numbered as in SEQ lD NO: 151.
106. The biosensor of claim 101, wherein the polypeptide comprises a mutant of ecTRX
that does not comprise a lysine.
107. The biosensor of claim 101, wherein the polypeptide further comprises a hexahistidine tag.
108. The biosensor of claim 101, comprising amino acids in the sequence set forth as any one of SEQ lD NOS: 24-41.
109. The biosensor of claim 101, which wherein the ligand comprises a hydrogen ion.
110. The biosensor of claim 101, which is a biosensor for pH, wherein the directly responsive fluorophore is pH-sensitive.

111. The biosensor of claim 110, wherein the fully excited emission intensity of the directly responsive fluorophore is different at a pH less than about 7.0 compared to a pH of 7.5.
112. The biosensor of claim 111, wherein the directly responsive fluorophore comprises a pH-sensitive fluorophore comprising fluorescein or a derivative thereof.
113. The biosensor of claim 110, wherein the directly responsive fluorophore transitions from a monoanion to a dianion at a pH that is less than 7.0 in an aqueous solution.
114. The biosensor of claim 75 or 99, wherein the directly responsive fluorophore is attached to the ligand-binding protein, the amino acid, or the polypeptide via a covalent bond.
115. The biosensor of claim 114, wherein the covalent bond comprises a disulfide bond, a thioester bond, a thioether bond, an ester bond, an amide bond, or a bond that has been formed by a click reaction.
116. The biosensor of claim 114, wherein the directly responsive fluorophore is attached to a cysteine or a lysine of the protein.
117. The biosensor of claim 114, wherein the indirectly responsive fluorophore is attached to the N-terminus or the C-terminus of the protein.
118. The biosensor of claim 114, wherein the indirectly responsive fluorophore is attached to the N-terminus or the C-terminus of the protein via a fluorophore attachment motif.
119. The biosensor of claim 118, wherein the fluorophore attachment motif comprises an amino acid or a polypeptide.
120. The biosensor of claim 119, wherein the polypeptide comprises amino acids in the sequence of .beta.Zif (SEQ ID NO: 42).

121. The biosensor of claim 119, wherein the polypeptide comprises a stretch of at least 50, 60, 70, 80, 90, or 100 amino acids in a sequence that is at least about 85%, 90%, 95%, or 99% identical to the amino acid sequence of E. coli thioredoxin (ecTRX; SEQ lD
NO: 151).
122. The biosensor of claim 76, wherein the indirectly responsive fluorophore is attached to the ligand-binding protein via a covalent bond.
123. The biosensor of claim 122, wherein the covalent bond comprises a disulfide bond, a thioester bond, a thioether bond, an ester bond, an amide bond, or a bond that has been formed by a click reaction.
125. The biosensor of claim 122, wherein the indirectly responsive fluorophore is attached to a cysteine or a lysine of the protein.
126. The biosensor of claim 122, wherein the indirectly responsive fluorophore is attached to the N-terminus or the C-terminus of the protein.
127. The biosensor of claim 122, wherein the indirectly responsive fluorophore is attached to the N-terminus or the C-terminus of the protein via a fluorophore attachment motif.
128. The biosensor of claim 127, wherein the fluorophore attachment motif comprises an amino acid or a polypeptide.
129. The biosensor of claim 128, wherein the polypeptide comprises amino acids in the sequence of .beta.Zif (SEQ lD NO: 42).
130. The biosensor of claim 138, wherein the polypeptide comprises a stretch of at least 50, 60, 70, 80, 90, or 100 amino acids in a sequence that is at least about 85%, 90%, 95%, or 99% identical to the amino acid sequence of E. coli thioredoxin (ecTRX; SEQ lD
NO: 151).
131. A method for assaying the level of a ligand in a subject, comprising contacting a biosensor according to claim 75 or 99 with a biological sample from the subject.

132. The method of claim 131, wherein the ligand comprises glucose, galactose, lactose, arabinose, ribose, maltose, lactate, urea, bicarbonate, phosphate, sulfate, chloride, fluoride, iodide, astatide, ununseptide, bromide, calcium, a hydrogen ion, a dipeptide, histidine, glutamine, glutamate, aspartate, or iron.
133. The method of claim 131, wherein the subject has or is suspected of having abnormal kidney function, abnormal adrenal gland function, diabetes, hypochloremia, bromism, hypothyroidism, hyperthyroidism, cretinism, depression, fatigue, obesity, a low basal body temperature, a goiter, a fibrocystic breast change, lactic acidosis, septic shock, carbon monoxide poisoning, asthma, a lung disease, respiratory insufficiency, Chronic Obstructive Pulmonary Disease (COPD), regional hypoperfusion, ischemia, severe anemia, cardiac arrest, heart failure, a tissue injury, thrombosis, or a metabolic disorder, diarrhea, shock, ethylene glycol poisoning, methanol poisoning, diabetic ketoacidosis, hypertension, Cushing syndrome, liver failure, cancer, or an infection.
134. The method of claim 131, wherein the biological sample comprises sweat, tear fluid, blood, serum, plasma, interstitial fluid, amniotic fluid, sputum, gastric lavage, skin oil, milk, fecal matter, emesis, bile, saliva, urine, mucous, semen, lymph, spinal fluid, synovial fluid, a cell lysate, venom, hemolymph, or a fluid obtained from a plant.
135. A method for assaying the level of ligand in an environmental sample, comprising contacting a biosensor according to claim 75 or 99 with the environmental sample.
136. The method of claim 135, wherein the environmental sample is from an environmental site that is suspected of being polluted.
137. The method of claim 135, wherein the environmental sample has been obtained or provided from an environmental substance, fluid, or surface.
138. The method of claim 137, wherein (a) the environmental substance comprises rock, soil, clay, sand, a meteorite, an asteroid, dust, plastic, metal, a mineral, a fossil, a sediment, or wood;
(b) the environmental surface comprises the surface of a satellite, a bike, a rocket, an automobile, a truck, a motorcycle, a yacht, a bus, or a plane, a tank, an armored personnel carrier, a transport truck, a jeep, a mobile artillery unit, a mobile antiaircraft unit, a minesweeper, a Mine-Resistant Ambush Protected (MRAP) vehicle, a lightweight tactical all-terrain vehicle, a high mobility multipurpose wheeled vehicle, a mobile multiple rocket launch system, an amphibious landing vehicle, a ship, a hovercraft, a submarine, a transport plane, a fighter jet, a helicopter, a rocket, or an Unmanned Arial Vehicle, a drone, a robot, a building, furniture, or an organism; or (c) the environmental fluid comprises marine water, well water, drinking well water, water at the bottom of well dug for petroleum extraction or exploration, melted ice water, pond water, aquarium water, pool water, lake water, mud, stream water, river water, brook water, waste water, treated waste water, reservoir water, rain water, or ground water.
139. A method for monitoring the level of a ligand, comprising periodically continuously detecting the level of the ligand, wherein detecting the level of the ligand comprises (a) providing or obtaining a sample;
(b) contacting the sample with a biosensor for the ligand according to claim 75 or 99, and (c) detecting a signal produced by the biosensor.
140. The method of claim 139, wherein the sample is provided or obtained from a subject or from a culture of microbial cells.
141. A method for constructing a biosensor, comprising:
(a) providing a ligand-binding protein;
(b) identifying at least one putative allosteric, endosteric, or peristeric site of the ligand-binding based a structure of the ligand-binding protein;
(e) mutating the ligand-binding protein to substitute an amino acid at the at least one putative allosteric, endosteric, or peristeric site of the second protein with a cysteine;
(f) conjugating a donor fluorophore or an acceptor fluorophore to the cysteine to produce single labeled biosensor;
(g) detecting whether there is a spectral shift or change in emission intensity of the single labeled biosensor upon ligand binding when the donor fluorophore or the acceptor fluorophore is fully excited; and (h) if a spectral shift or change in emission intensity is detected in (g), attaching a donor fluorophore to the second protein if an acceptor fluorophore is attached to the cysteine, and attaching an acceptor fluorophore to the second protein if an acceptor fluorophore is attached to the cysteine.
142. The method of claim 141, wherein the ligand-binding protein has been identified by (i) selecting a first protein having a known amino acid sequence (seed sequence), wherein the first protein is a ligand-binding protein;
(ii) identifying a second protein having an amino acid sequence (hit sequence) with at least 15% sequence identity to the seed sequence;
(iii) aligning the seed amino acid sequence and the hit sequence, and comparing the hit sequence with the seed sequence at positions of the seed sequence that correspond to at least 5 primary complementary surface (PCS) amino acids, wherein each of the at least 5 PCS amino acids has a hydrogen bond interaction or a van der Waals interaction with ligand when ligand is bound to the first protein; and (iv) identifying the second protein to be a ligand-binding protein if the hit sequence comprises at least 5 amino acids that are consistent with the PCS.
143. The method of claim 141, wherein the spectral shift comprises a monochromatic fluorescence intensity change or a dichromatic spectral shift.
144. A method of converting a biosensor that shows a monochromatic response upon ligand binding into a biosensor with a dichromatic response upon ligand binding, the method comprising (a) selecting a biosensor that exhibits a monochromatic response upon ligand binding, wherein the biosensor comprises a ligand-binding protein and a first reporter group;
and (b) attaching a second reporter group to the biosensor, wherein the second reporter group has (i) an excitation spectrum that overlaps with the emission spectrum of the first reporter group; or (ii) an emission spectrum that overlaps with the excitation spectrum of the first reporter group.

145. The method of claim 144, wherein the second reporter group is within about 100 angstroms (.ANG.) of the first reporter group regardless of whether ligand is bound to the biosensor.
146. The method of claim 144, wherein when the ligand is bound to the biosensor, the average distance between the first reporter group and the second reporter group changes by less than about 1 .ANG. compared to when ligand is not bound to the ligand-binding protein.
147. A method of converting a biosensor that shows a monochromatic response upon ligand binding into a biosensor with a dichromatic response upon ligand binding, the method comprising (a) selecting a biosensor that exhibits a monochromatic response upon ligand binding, wherein the biosensor comprises a ligand-binding fluorescent protein;
and (b) attaching an acceptor fluorophore or a donor fluorophore to the biosensor, wherein (i) the acceptor fluorophore has an excitation spectrum that overlaps with the emission spectrum of the fluorescent protein; or (ii) the donor fluorophore has an emission spectrum that overlaps with the excitation spectrum of the fluorescent protein.
148. A method of increasing a dichromatic response of a biosensor to ligand binding, the method comprising (a) selecting a biosensor that exhibits a dichromatic response upon ligand binding, wherein the biosensor comprises a ligand-binding protein and a first reporter group; and (b) attaching a second reporter group to the biosensor, wherein the second reporter group has (i) an excitation spectrum that overlaps with the emission spectrum of the first reporter group; or (ii) an emission spectrum that overlaps with the excitation spectrum of the first reporter group.
149. A method of converting a biosensor that shows a monochromatic response upon ligand binding into a biosensor with a dichromatic response upon ligand binding, the method comprising (a) selecting a biosensor that exhibits a monochromatic response upon ligand binding, wherein said biosensor comprises an amino acid or a polypeptide and a first reporter group, wherein the first reporter group comprises a chemoresponsive fluorophore; and (b) attaching a second reporter group to said biosensor, wherein said second reporter group has (i) an excitation spectrum that overlaps with the emission spectrum of said first reporter group; or (ii) an emission spectrum that overlaps with the excitation spectrum of said first reporter group.
150. A method of increasing a dichromatic response of a biosensor to ligand binding, the method comprising (a) selecting a biosensor that exhibits a dichromatic response upon ligand binding, wherein said biosensor comprises an amino acid or a polypeptide and a first reporter group, wherein the first reporter group comprises a chemoresponsive fluorophore; and (b) attaching a second reporter group to said biosensor, wherein said second reporter group has (i) an excitation spectrum that overlaps with the emission spectrum of said first reporter group; or (ii) an emission spectrum that overlaps with the excitation spectrum of said first reporter group.
151. A method for monitoring the level of a ligand, comprising periodically detecting the level of the ligand, wherein detecting the level of the ligand comprises (a) providing or obtaining a sample;
(b) contacting the sample with a biosensor for glucose according to claim 75 or 99 under conditions such that the ligand-binding protein of the biosensor binds to the ligand, and (c) detecting a signal produced by the biosensor.
152. A method for monitoring the level of a ligand, comprising continuously detecting the level of the ligand, wherein detecting the level of the ligand comprises (a) providing or obtaining a sample;
(b) contacting the sample with a biosensor for the ligand according to claim 75 or 99 under conditions such that the ligand-binding protein of the biosensor binds to the ligand, and (c) detecting a ratiometric signal produced by the biosensor.

153. A method for monitoring the level of ligand in a subject, comprising (a) administering a biosensor according to claim 75 or 99 or a device comprising a biosensor according to claim 75 or 99 to the subject, wherein after administration the biosensor is in contact with a bodily fluid or surface of the subject, and (b) detecting (i) a signal produced by a reporter group of the biosensor continuously or repeatedly at intervals less than about 30 minutes apart, and/or (ii) whether a signal is produced by a reporter group of the biosensor continuously or repeatedly at intervals less than about 30 minutes apart.
154. A device comprising a biosensor according to claim 75 or 99.
155. The device of claim 154, wherein the biosensor is attached to a surface or matrix of the device, wherein the surface or matrix comprises a polymer.
156. The device of claim 154, wherein the biosensor is attached to a surface or matrix of the device, wherein the surface or matrix comprises cellulose.
157. The device of claim 154, which comprises an optode, a dermal patch, a contact lense, a bead that is suitable for subcutaneous administration, or a tattoo composition.