WO2016156809A1 - Atp sensor - Google Patents

Atp sensor Download PDF

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Publication number
WO2016156809A1
WO2016156809A1 PCT/GB2016/050818 GB2016050818W WO2016156809A1 WO 2016156809 A1 WO2016156809 A1 WO 2016156809A1 GB 2016050818 W GB2016050818 W GB 2016050818W WO 2016156809 A1 WO2016156809 A1 WO 2016156809A1
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atp
seq
binding molecule
atp binding
suitably
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French (fr)
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Martin R. WEBB
Renee VANCRAENENBROECK
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Medical Research Council
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Medical Research Council
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites

Definitions

  • BACKGROUND TO THE INVENTION ATP is an intracellular energy source, for example involved in active transport, cell motility and biosynthesis. It is also an important extracellular signalling agent in neurotransmission (Burnstock 2012) and inflammation (Idzko, Ferrari et al. 2014).
  • ATP is generated through several pathways such as glycolysis, the Krebs cycle and oxidative phosphorylation. This makes it an important assay target and monitoring.
  • ATP production is widely used to measure enzyme activity in biochemical and cell- based applications.
  • Various ATP assays are known in the art.
  • Coupled-enzyme assays including the luciferase-luciferin system (Patergnani, Baldassari et al. 2014), require several reagents, which is a drawback.
  • the present invention seeks to overcome problem(s) associated with the art.
  • a fluorescent, reagentless biosensor for ATP is described.
  • biosensors for a target molecule are a single molecular species that consists minimally of a recognition element and a reporter.
  • the recognition element is a protein that interacts with the target, ATP, namely an ANL superfamily protein.
  • the ANL superfamily protein malonyl-coenzymeA synthetase from Rhodopseudom onas palustris (RpMatB) is used. Amino acid sequence derived from this protein is coupled covalently to reporter fluorophore(s) to give a fluorescence change on ATP binding.
  • a fluorescent reagentless biosensor for ATP was developed based on malonyl-coenzyme A synthetase from Rhodopseudom onas palustris (RpMatB) as the protein scaffold and recognition element.
  • RpMatB Rhodopseudom onas palustris
  • two 5- iodoacetamidotetramethylrhodamines were covalently bound to RpMatB to provide the readout.
  • This adduct couples ATP binding to a 3.7-fold increase in fluorescence intensity with excitation at 553 nm and emission at 575 nm. It has micromolar sensitivity for ATP and is highly selective for ATP relative to ADP. Its ability to monitor ATP production was demonstrated in a steady-state kinetic assay in which ATP is a product.
  • the invention provides an ATP binding molecule comprising a polypeptide which undergoes a conformational change from a first conformation to a second conformation upon binding of ATP, said polypeptide comprising amino acid sequence corresponding to at least amino acids 5 to 497 of SEQ ID NO:i,
  • polypeptide having at least 21% sequence identity to SEQ ID NO:i,
  • polypeptide having a value of RMSD ⁇ 4 A relative to RpMatB in the ATP-bound conformation
  • polypeptide comprises a cysteine residue for attachment of at least one reporter moiety, and comprises at least one reporter moiety attached thereto, wherein said cysteine residue for attachment of at least one reporter moiety is positioned such that said reporter moiety undergoes a change in fluorescence upon changing from said first conformation to said second conformation.
  • the RMSD is calculated using the PyMOL program.
  • the RMSD is calculated using the "align" command in the PyMOL program.
  • RpMatB in the ATP-bound conformation is taken as PDB number '4FUT'.
  • said polypeptide has a value of RMSD ⁇ 4 A relative to PDB number '4FUT' (i.e. RpMatB in the ATP-bound conformation).
  • sequence identity to SEQ ID NO:i is for the amino acid residues corresponding to those shown in column II of table A. Column II of table A corresponds to 'exposed' residues.
  • the polypeptide comprises a first cysteine residue at a position corresponding to a position selected from R286 , A282, D283, H285, E287, S289, A290, K385 , L383, G384, 1386 and D287 of SEQ ID NO: 1,
  • polypeptide comprises a second cysteine residue at a position corresponding to a position selected from
  • polypeptide comprises a first cysteine residue at a position corresponding to a position selected from R286 and K385 of SEQ ID NO: 1,
  • polypeptide comprises a second cysteine residue at a position corresponding to a position selected from Q457, G461 and K470 of SEQ ID NO: 1.
  • polypeptide comprises a first cysteine residue at a position corresponding to position R286 of SEQ ID NO: 1,
  • polypeptide comprises a second cysteine residue at a position corresponding to position G461 of SEQ ID NO: 1.
  • polypeptide comprises a first cysteine residue at a position corresponding to position K385 of SEQ ID NO: 1,
  • polypeptide comprises a second cysteine residue at a position corresponding to position K470 of SEQ ID NO: 1.
  • polypeptide comprises a first cysteine residue at a position corresponding to position R286 of SEQ ID NO: 1,
  • polypeptide comprises a second cysteine residue at a position corresponding to position Q457 of SEQ ID NO: 1.
  • said molecule comprises the amino acid sequence of SEQ ID NO: 4, SEQ ID NO:5 or SEQ ID NO:6.
  • said molecule further comprises at least two tetramethylrhodamine moieties attached thereto.
  • each of said at least two tetramethylrhodamine moieties is independently selected from the group consisting of 5-tetramethylrhodamine and 6-tetramethylrhodamine.
  • the invention relates to an ATP binding molecule as described above wherein said cysteine residue for attachment of a reporter moiety is at a position corresponding to a position selected from E439 , I403, P433, G438, G440, N492 , K491, V493, R495, E496 and T497 of SEQ ID NO: 1.
  • cysteine residue for attachment of a reporter moiety is at a position corresponding to E439 or N492 of SEQ ID NO: 1.
  • said molecule comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO:3.
  • said molecule further comprises at least one diethylaminocoumarin moiety attached thereto.
  • said diethylaminocoumarin moiety is independently selected from the group consisting of (N-[2-(i-maleimidyl)ethyl]-7-diethylaminocoumarin-3-carboxamide and N-[2-(iodoacetamido)ethyl]-7-diethylaminocoumarin-3-carboxamide).
  • the invention relates to an ATP binding molecule as described above wherein said polypeptide comprises an amino acid other than Threonine at the position corresponding to T167 of SEQ ID NO: 1.
  • said polypeptide comprises alanine or serine at the position corresponding to T167 of SEQ ID NO: 1, preferably alanine.
  • the invention relates to an ATP binding molecule as described above wherein said polypeptide comprises an amino acid other than Serine at the position corresponding to S170 of SEQ ID NO: 1.
  • said polypeptide comprises alanine at the position corresponding to S170 of SEQ ID NO: 1.
  • the invention relates to an ATP binding molecule as described above wherein said polypeptide comprises an amino acid other than Threonine at the position corresponding to T303 of SEQ ID NO: 1.
  • said polypeptide comprises alanine or serine at the position corresponding to T303 of SEQ ID NO: 1, preferably alanine.
  • the invention relates to an ATP binding molecule as described above wherein said polypeptide comprises an amino acid other than cysteine at the position corresponding to C106 of SEQ ID NO: l.
  • the invention relates to an ATP binding molecule as described above wherein said polypeptide comprises alanine at the position corresponding to C106 of SEQ ID NO: ⁇ . (CIO 6 A)
  • the invention relates to an ATP binding molecule as described above wherein said polypeptide comprises an active site mutation such that the polypeptide is catalytically inactive for ATP hydrolysis.
  • the invention relates to an ATP binding molecule as described above wherein said active site mutation comprises an amino acid other than lysine at the position corresponding to K488 of SEQ ID NO: 1.
  • the invention relates to an ATP binding molecule as described above wherein said polypeptide comprises alanine at the position corresponding to K488 of SEQ ID NO: 1. (K488A)
  • the invention relates to an ATP binding molecule as described above further comprising the sequence of SEQ ID NO: 8.
  • the invention relates to a nucleic acid having a nucleotide sequence encoding the polypeptide portion of an ATP binding molecule as described above.
  • the invention relates to a method for monitoring changes in ATP concentration in a sample comprising contacting said sample with an ATP binding molecule as described above and determining changes in conformation of said ATP binding molecule, wherein changes in conformation of said ADP binding molecule indicate changes in the concentration of ATP in said sample.
  • the conformation of said ATP binding molecule is monitored by measurement of changes in fluorescence of a fluorophore comprised by said ATP binding molecule.
  • the sample comprises divalent Magnesium ion (Mg 2+ ).
  • the invention relates to use of an ATP binding molecule as described above in the determination of ATP concentration in a sample.
  • the invention relates to an ATP binding molecule comprising a polypeptide which undergoes a conformational change from a first conformation to a second conformation upon binding of ATP, said polypeptide comprising amino acid sequence corresponding to at least amino acids 5 to 497 of SEQ ID NO:i,
  • polypeptide having at least 34% sequence similarity to SEQ ID NO:i,
  • polypeptide comprises a cysteine residue for attachment of at least one reporter moiety, and comprises at least one reporter moiety attached thereto, wherein said cysteine residue for attachment of at least one reporter moiety is positioned such that said reporter moiety undergoes a change in fluorescence upon changing from said first conformation to said second conformation.
  • the invention relates to an ATP binding molecule comprising a polypeptide which undergoes a conformational change from a first conformation to a second conformation upon binding of ATP, said polypeptide comprising amino acid sequence corresponding to at least amino acids 5 to 497 of SEQ ID NO:i,
  • polypeptide having at least 21% sequence identity to SEQ ID NO:i,
  • polypeptide comprises a cysteine residue for attachment of at least one reporter moiety, and comprises at least one reporter moiety attached thereto, wherein said cysteine residue for attachment of at least one reporter moiety is positioned such that said reporter moiety undergoes a change in fluorescence upon changing from said first conformation to said second conformation.
  • the invention provides a single solution to the problem of provision of an ATP biosensor.
  • the biosensor is singly labelled.
  • the biosensor is doubly labelled.
  • the single dye molecule attached to the biosensor interacts with the protein, and this interaction affects the signal such as fluorescence. Binding of ATP changes the conformation of the biosensor, and thus alters the interaction the dye molecule with the polypeptide. This ATP bound states exhibits a difference in reporter activity such as fluorescence and therefore serves to read out the event of ATP binding.
  • the two dye molecules attached to the biosensor interact with one another, for example via dye stacking.
  • the single label or dual labelling approaches both rely on a signal such as fluorescence signal due to the closing of the "lid domain" on ATP binding.
  • the single label detects the movement through interactions with the protein surface.
  • the dual (e.g. rhodamine) label detects the relative position of the two labels, which changes on the closure.
  • a doubly labelled biosensor of the invention is especially suitable.
  • the biosensor comprises a first label and a second label which can exhibit molecular stacking and wherein the molecular stacking is altered on changing from one conformation to the other.
  • the first and second labels can exhibit molecular stacking either (a) in the first conformation but not in the second conformation, or (b) in the second conformation but not in the first conformation.
  • the first and second labels exhibit molecular stacking in the first conformation.
  • the first and second labels exhibit molecular stacking in the second conformation.
  • a doubly labelled biosensor suitably uses rhodamine dye molecules. These embodiments of the invention can produce the largest signal.
  • dyes such as rhodamine can exhibit superior stability during fluorescence measurements, such as greater stability under irradiation.
  • rhodamine is more photostable than coumarins.
  • rhodamine has a longer wavelength excitation, often making it easier to use.
  • the dye used in the biosensors of the invention is rhodamine.
  • sensors of the invention may exist in two conformations. It is thought that these conformations may reflect monomelic and dimeric forms of the protein. It appears that the monomeric form is the most effective at ATP binding. Nevertheless, it is clear from the practical experiments provided in the application that whether or not this theory is correct, the sensors perform very well regardless of the precise description of their monomeric/dimeric forms.
  • the polypeptide of a sensor of the invention may comprise a structural homologue of RpMatB, such as an ANL superfamily protein.
  • the term 'ANL superfamily' is well known in the art.
  • the ANL superfamily of adenylating enzymes contains acyl- and aryl-CoA synthetases, firefly luciferase, and the adenylation domains of the modular Non-Ribosomal Peptide Synthetases (NRPSs).
  • NRPSs Non-Ribosomal Peptide Synthetases
  • Members of this family catalyse two partial reactions, the initial adenylation of a carboxylate to form an acyl-AMP intermediate, followed by a second partial reaction, most commonly, the formation of a thioester. This is described in more detail in the art, such as in Gulick 2009 (ACS Chem Biol. 2009 October 16; 4(10): 811-827. doi:io.i02i/cb900i56h), which is incorporated herein by reference specifically for the information relating to the ANL superfamily.
  • the Pfam database is a large collection of protein families, each represented by multiple sequence alignments and hidden Markov models (HMMs).
  • HMMs hidden Markov models
  • the release number of the database referred to is Pfam 27.0 (March 2013, 14831 families).
  • This superfamily consists of enzymes including luciferase, long chain fatty acid Co-A ligase, acetyl-CoA synthetase and various other closely-related synthetases as well as a plant auxin-responsive promoter family.
  • the name ANL derives from from three of the subfamilies - Acyl-CoA synthetases, the NRPS adenylation domains, and the Luciferase enzymes [Gulick AM;, ACS Chem Biol.
  • the "lid” domain upon which the conformational change on ATP binding is based, is common to ANL superfamily proteins.
  • ANL superfamily members have two partial reactions.
  • the invention relates to ATP sensors which comprise an ANL superfamily protein.
  • said ANL superfamily protein comprises variants of RpMatB having amino acid substitutions as described, or comprises RpMatB as exemplified.
  • sequence identity/ similarity is assesses along the whole length of the amino acid sequence present in the polypeptide component of the ATP sensor, unless otherwise specified.
  • RMSD Root Mean Square Deviation
  • RMSD is calculated between structures (rather than sequences).
  • the RMSD can be used to compare protein three-dimensional structures.
  • the RMSD is 0 for identical structures, and its value increases as the two structures become more different.
  • RMSD values are considered as reliable indicators of variability when applied to very similar proteins.
  • a value of RMSD ⁇ 4 A indicates significant structural similarities.
  • PyMOL used in Example 12 calculates it after cycles of refinement in order to reject structural outliers found during the fit - at least when you use the "align" command.
  • Another program, SuperPose does not follow this procedure. Its RMSD is usually higher than the one calculated via PyMOL (it takes more atoms into account). The skilled worker will be aware of these differences.
  • the RMSD is calculated using the PyMOL program.
  • RMSD values mentioned herein are calculated using the PyMOL program.
  • the PyMOL program is available for example from htt : // w . py moi . org/, or from Schrodinger, 101 SW Main Street, Suite 1300, Portland, OR 97204, USA.
  • RMSD is calculated using the "align” command in the PyMOL program.
  • RMSD values mentioned herein are calculated using the "align” command in the PyMOL program.
  • RpMatB exists in multiple conformations (see below), so it is important to calculate the RMSD values for the structure pairs that have the same conformation.
  • RMSD values mentioned herein are for the ATP bound conformation(s).
  • the reference structure is RpMatB in the ATP-bound conformation.
  • the polypeptide component of the ATP sensor of the invention is comprises an ANL superfamily protein.
  • said polypeptide comprises a value of RMSD ⁇ 4 A relative to RpMatB; more suitably said polypeptide comprises a value of RMSD ⁇ 3 A relative to RpMatB.
  • RpMatB was chosen as the exemplary recognition element of the ATP sensors described because of several properties, including high yield expression and purification, good stability and high affinity and selectivity for ATP.
  • RpMatB belongs to the AMP-forming acyl-coenzymeA synthetase family (PF00501 (Finn, Bateman et al. 2014)) and the ANL superfamily containing acyl- and aryl-coenzymeA synthetases, the adenylation domains of nonribosomal peptide synthetases and firefly luciferase (Gulick 2009).
  • RpMatB catalyzes the conversion of malonate and coenzymeA to malonyl- coenzymeA via a ping-pong mechanism consuming ATP through a malonyl-AMP intermediate. Its products are AMP, pyrophosphate and malonyl-coenzymeA.
  • RpMatB has been crystallized in two conformations, that is an open form of the apoprotein and a closed form with MgATP bound (Crosby, Rank et al. 2012) ( Figure 1).
  • the ligand-binding pocket is between the N- and C-terminal lobes.
  • the C-terminal lobe rotates ⁇ 20° to close the binding cleft.
  • This conformational change was used to create a series of rationally designed RpMatB mutants with cysteine point mutations of surface amino acid residues in order to incorporate thiol-reactive fluorophores.
  • the exemplary ATP biosensor described herein is an adduct of RpMatB and tetramethylrhodamine, which specifically responds to ATP with a maximum 3.7-fold fluorescence increase. Its sensitivity lies in the micromolar range. Its ability to monitor ATP production was demonstrated with a steady-state kinetic assay to measure the time course of enzymatic ATP production.
  • polypeptide of a sensor of the invention comprises amino acid sequence corresponding to RpMatB amino acid sequence, comprising substitutions as described.
  • w ild-type RpMat se quence accession number: Genbank CAE25665.1
  • polypeptide components of the molecules of the invention are based on ANL superfamily polypeptide sequences such as the exemplary RpMatB sequence.
  • amino acid addresses given in the application correspond to the numbering of the RpMatB reference sequence of SEQ ID NO:i.
  • truncated or extended forms of RpMatB are used as polypeptides in molecules of the invention (e.g. where a 6his tag is added or where a section of the polypeptide is deleted) then the amino acid numbering should be treated as corresponding to the equivalent section of the full length RpMatB reference sequence and not as an 'absolute' or rigidly inflexible numeric address.
  • the exemplary biosensor polypeptide RpMatB has been studied in detail and each residue has been classified as set out in Table A below. The classification is as follows, making use of information on surface exposure:
  • Buried Residues in the core of the structure where mutations are likely to affect the function of the sensor.
  • Residues marked with an asterisk (*) designate active site residues.
  • Active site residues may suitably be specifically mutated in order to optimise the sensor functions (such as to impair or eliminate ATP hydrolysis such as for example mutating K488). These are discussed in more detail in the text.
  • ASA relative accessible surface area
  • ASA is at least 40% of its nominal maximum area.
  • a residue is defined as buried if its relative ASA is less than 10% of its nominal maximum area.
  • Residues marked with asterisk (*) designate active site residues (within 6 A of ATP and Mg 2+ ).
  • residues in the polypeptide part of an ATP sensor molecule of the invention have at least 90% sequence identity to RpMatB (SEQ ID NO:i). Suitably any differences are conservative substitutions.
  • residues in the polypeptide part of an ATP sensor molecule of the invention have 100% similarity to RpMatB (SEQ ID NO:i). More suitably residues shown as 'buried' in table A are not mutated.
  • residues in the polypeptide part of an ATP sensor molecule of the invention which correspond to RpMatB residues shown as 'buried' in table A are not mutated relative to RpMatB (SEQ ID NO:i).
  • residues in the polypeptide part of an ATP sensor molecule of the invention shown as 'buried' in table A comprise the same residue as at the corresponding position in RpMatB (SEQ ID NO:i).
  • the polypeptide component of the ATP sensor molecule of the invention suitably comprises amino acid sequence having 100% sequence identity to those residues shown as 'buried' in table A.
  • residues shown as 'intermediate' in table A may be mutated.
  • residues in the polypeptide part of an ATP sensor molecule of the invention which correspond to RpMatB residues shown as 'intermediate' in table A may be mutated relative to RpMatB (SEQ ID NO:i).
  • residues in the polypeptide part of an ATP sensor molecule of the invention shown as 'intermediate' in table A may comprise a different residue (or no residue) from the corresponding position in RpMatB (SEQ ID NO:i).
  • a biosensor of the invention has at least 60% sequence identity to SEQ ID NO: 1.
  • the polypeptide component of the ATP sensor of the invention suitably comprises amino acid sequence having at least 60% sequence identity to those residues shown as 'intermediate' in table A.
  • the polypeptide component of the ATP sensor of the invention suitably comprises amino acid sequence having at least 68% sequence identity to those residues shown as 'intermediate' in table A, suitably least 70% sequence identity, suitably least 74% sequence identity, suitably least 78% sequence identity, suitably least 82% sequence identity, suitably least 86% sequence identity, suitably least 90% sequence identity, suitably least 94% sequence identity, suitably least 98% sequence identity to those residues shown as 'intermediate' in table A.
  • these 'intermediate' residues are in fact partially buried.
  • these 'intermediate' residues are only mutated by substitution with a conservative residue relative to RpMatB.
  • the non- identical residues noted above comprise only conservative substitutions relative to the corresponding residue in RpMatB.
  • sequence similarity takes account of sequence identity and also takes account of conservative substitutions (i.e. non-identical residues but where the residue is similar or conserved compared to the original residue). Assessing sequence similarity is well known in the art. Examples are provided in the examples section. In case any further guidance is needed, suitably the following parameters are used in the algorithm for calculating sequence similarity: BLOSUM62 matrix, gap penalty 10.0, gapextend penalty 0.5; more suitably BLOSUM62 matrix, gapopen 10.0, gapextend 0.5, endopen 10.0, endextend 0.5, pairwise alignment.
  • the polypeptide component of the ATP sensor of the invention has at least 34% sequence similarity to SEQ ID NO: 1, suitably at least 38% sequence similarity, suitably at least 40% sequence similarity, suitably at least 50% sequence similarity, suitably at least 60% sequence similarity, suitably at least 65% sequence similarity, suitably at least 70% sequence similarity, suitably at least 75% sequence similarity, suitably at least 80% sequence similarity, suitably at least 85% sequence similarity, suitably at least 90% sequence similarity, suitably at least 95% sequence similarity, suitably at least 98% sequence similarity, suitably at least 99% sequence similarity, most suitably 100% similarity to SEQ ID NO: 1.
  • residues shown as 'intermediate' in table A are not mutated.
  • residues in the polypeptide part of an ATP sensor of the invention which correspond to residues shown as 'intermediate' in table A are not mutated relative to RpMatB (SEQ ID NO:i).
  • residues in the polypeptide part of an ATP sensor of the invention shown as 'intermediate' in table A comprise the same residue as at the corresponding position in RpMatB (SEQ ID NO:i).
  • residues shown as 'exposed' in table A may be mutated.
  • residues in the polypeptide part of an ATP sensor of the invention which correspond to residues shown as 'exposed' in table A may be mutated relative to RpMatB (SEQ ID NO:i).
  • residues in the polypeptide part of an ATP sensor of the invention shown as 'exposed' in table A may comprise a different residue (or no residue) from the corresponding position in RpMatB (SEQ ID NO:i).
  • the polypeptide component of the ATP sensor of the invention suitably comprises amino acid sequence having at least 21% sequence identity to those residues shown as 'exposed' in table A, suitably at least 30% sequence identity, suitably at least 40% sequence identity, suitably at least 50% sequence identity, suitably at least 60% sequence identity, suitably at least 65% sequence identity, suitably at least 70% sequence identity, suitably at least 75% sequence identity, suitably at least 80% sequence identity, suitably at least 85% sequence identity, suitably at least 90% sequence identity, suitably at least 95% sequence identity, suitably at least 98% sequence identity, suitably at least 99% sequence identity, most suitably 100% identity to those residues shown as 'exposed' in table A.
  • a near neighbour of the specified amino acid may mean an adjacent amino acid i.e. the amino acid before or the amino acid after the one specified.
  • neighbouring amino acid may be used.
  • a neighbouring amino acid may refer to an amino acid two residues either side of the residue specified, for example if the residue specified is 398 then residues 400 and 396 would be considered neighbouring amino acids. For example, if amino acid 398 was specified, then residues 399 and 397 would be considered near neighbours.
  • a further neighbour of the amino acid may be specified, for example, if amino acid 398 is taught for attachment then a further neighbour might be three amino acid residues away such as residue 401 or residue 395. More distant amino acids may be used if desired. In all cases, it is advisable to check the performance of the sensor using the assays as taught herein.
  • an amino acid which is "in the vicinity" of a specified amino acid is one which is present in a physically adjacent 3-dimensional space.
  • an amino acid specified on a part of an a helix will have a neighbouring amino acid in the vicinity such as the residue at the same position on the next turn of that a helix.
  • amino acid 457 is on one side of an a helical section of the protein; thus amino acid 461 is a neighbouring amino acid in the vicinity of amino acid 457 since it is on the corresponding side of the next turn of the same a helix.
  • an amino acid which might seem "distant" in terms of the number of intervening residues may actually be a neighbouring amino acid in the vicinity of the specified amino acid if it is close in 3-dimensional space.
  • Amino acid residues close in space to those exemplified for labelling may also be suitable for labelling. Examples of surface accessible residues & neighbouring amino acids are now discussed to aid understanding.
  • Table 2 shows examples of amino acids which are neighbouring in sequence or in space. In the latter case, this represents amino acids in a similar location on the next turn of a helix, on the same side of a beta-sheet or on an adjacent sheet of a beta-plate.
  • each reporter moiety (such as fluorophore) is attached to the polypeptide via an amino acid residue corresponding to one or more of those listed in the above table.
  • these exemplary sequences may have additions/deletions (e.g. N- or C- terminal truncations) or other substitutions as described.
  • SEQ ID NO: 7 shows exemplary sequence annotated to illustrate exemplary substitutions which may be used in the invention.
  • these exemplary sequences may further comprise an N-terminal addition MSYYHHHHHH DYDIPTSENL YFQGAS (SEP ID NO: 8 ) added directly before the first Methionine of the sequences above.
  • This N-terminal addition comprises a 6His tag useful in purification.
  • an ANL superfamily polypeptide for use as an ATP sensor molecule or ATP binding molecule.
  • Such an ANL superfamily polypeptide may be full length (i.e. comprising all 503 amino acid residues corresponding to SEQ ID NO:i (whether or not substitutions relative to SEQ ID NO:i are made in the particular amino acids present)) or truncated.
  • truncated forms are those which lack a small number of amino acid residues from the N- or C- terminus of the polypeptide relative to wild type.
  • a small number is 10 or fewer.
  • ANL superfamily proteins such as RpMatB have flexible C- and N- terminal ends. We teach that small truncations might be made at either or both ends. Suitably some or all of the amino acids from these flexible sections may be deleted without adversely affecting the remaining structure and hence retaining sensor function.
  • N-terminal amino acids which may be deleted include those corresponding to Ml, N2, A3, N4 of SEQ ID NO: 1.
  • C-terminal amino acids which may be deleted include those corresponding to E498, K499, D500, 1501, Y502, K503 of SEQ ID NO: 1.
  • small insertions or deletions may be made in the protein without disrupting function, in particular insertions or deletions are suitably not made in the region of the reporter/dye attachment points, nor in the ATP binding section of the protein.
  • insertions or deletions are suitably not made in secondary structure elements of the polypeptide such as alpha helices or beta sheets.
  • insertions/deletions are not made in dual labelled polypeptides in the section of the polypeptide between the dye attachment points, so as to preserve the dye spacing.
  • Suitably tags may be placed at the extreme C-terminus or the extreme N-terminus of the sensor molecule.
  • tags are placed at the N-terminus of the protein.
  • one tag per protein molecule is used.
  • Multiple tags per protein molecule may be used if desired, including multiple copies of the same tag or two or more different tags, for example it may be desirable to use a 6 His-tag for purification and in addition to use a Myc-tag for detection.
  • Tags may be removed from the sensor protein, for example by proteolytic cleavage, or may be retained on the sensor protein during use.
  • a hexahistidine tag (6his) may be added to the polypeptide part of the ATP binding molecule of the invention to simplify purification; most suitably a N-terminal hexahistidine tag is used. 6 His is a particularly useful tag for purification on nickel substrates. However, any suitable tag known in the art may be used. Alternatively, the sensor molecule of the invention may be tagless. Tagless purification (if needed) is well known in the art.
  • ANL superfamily proteins may have catalytic activity.
  • RpMatB may consume ATP when carrying out its enzymatic activity.
  • RpMatB has no significant ATPase activity (forming ADP and Pi).
  • the reaction for RpMatB is:
  • sensors of the invention with an intact catalytic activity.
  • the sensor molecules of the invention are catalytically inactive.
  • the sensor molecules of the invention do not comprise ATPase activity.
  • the sensor molecules of the invention are catalytically inactive for ATP consumption.
  • the sensor molecule may be catalytically inactivated by mutation of the active site.
  • the sensor of the invention is mutated to render it catalytically inactive.
  • the sensor comprises an ANL superfamily member such as RpMatB, suitably a K488 mutation is present.
  • the amino acid corresponding to the wild type K488 is changed to any amino acid other than K.
  • K488V may be used.
  • K488A may be used.
  • the sensor of the invention comprises K488X 1 , wherein X 1 is not K.
  • the sensor of the invention comprises K488V.
  • the sensor of the invention comprises K488A.
  • the conjugation of dye to the polypeptide of interest may target naturally occurring cysteine residues. To this end, it may be useful to remove unwanted cysteine residues by a process of mutation in order to eliminate background signal.
  • cysteine 106 of the wild type protein is mutated to be other than cysteine.
  • the sensor of the invention comprises a C106X 2 mutation, wherein X 2 is not C.
  • the sensor of the invention comprises a C106A mutation. Background signal from C106 may be as high as 6%. Therefore, C106X 2 (where X 2 is not C) mutants such as C106A provide the advantage of eliminating this background signal.
  • a sensor which is wild type at position 106 i.e. comprising C106 may still be used - in this case the values collected should be adjusted for any background signal as necessary.
  • polypeptide components of the ATP sensor molecules of the invention may be produced by standard recombinant techniques, such as creating a nucleic acid encoding the amino acid sequence of the polypeptide, and then expressing the polypeptide in a host such as E.coli. Alternatively an in vitro translation may be used. Alternatively the polypeptide itself may be chemically synthesised.
  • polypeptide(s) may be purified by any suitable method known in the art, such as 6His tagging the protein then purification using Ni-NTA beads.
  • any suitable technique may be used such as site directed mutagenesis.
  • mutant PCR primers or oligonucleotides containing the desired nucleotide sequence may be annealed to a template and ligated, extended or amplified to produce a mutated nucleotide sequence encoding the desired substitution.
  • the desired nucleotide sequence may be synthesised chemically. Exemplary techniques are presented in the Examples below. REPORTER MOIETIES / DYES
  • the ATP binding molecules of the invention comprise at least one reporter moiety attached thereto.
  • the reporter moiety may be any suitable chemical group or structure capable of reading out change(s) in the conformation of said ATP binding molecule.
  • the reporter moiety comprises one or more fluorophore(s) such as coumarin or rhodamine.
  • Reporter moieties used in the invention can give various signals, but preferred labels are luminescent labels.
  • Luminescent labels include both fluorescent labels and phosphorescent labels.
  • electrochemical labels could be used wherein alteration in the environment of the labels will give rise to a change in redox state. Such a change may be detected using an electrode.
  • fluorescent labels which may be excited to fluoresce upon exposure to certain wavelengths of light are used.
  • the fluorescent label can be selected from the group consisting of rhodamines, cyanines, pyrenes and derivatives thereof.
  • Preferred fluorescent fluorophores are based on a xanthene nucleus, which can readily undergo stacking to form dimers. Especially suitable are rhodamine fluorophores.
  • the reporter moiety comprises any usable fluorescent label. Fluorescent labels with environmentally sensitive fluorescence are most suitable. When a cysteine is the site of attachment, then the moiety needs thiol-reactivity for attachment. In other embodiments, an amine-sensitive label on a non-Cys amino acid may be employed.
  • reporter moieties may be those that can exhibit molecular stacking, which will thus include aromatic rings. These include the rhodamine labels. In other embodiments labels which do not stack may be used, such as coumarin labels.
  • Dye stacking is a non-covalent interaction between two chromophores having planar aromatic rings, and it occurs when the rings are separated by a distance that is short enough to allow them to interact e.g. to form dimers or trimers.
  • the detectable signal of the stacked molecules is different from that of the unstacked molecules (e.g. stacking can cause quenching of signals, and so stacked chromophores will typically show a decreased fluorescence signal intensity relative to the individual unstacked chromophores), and this difference can be used to detect the presence or absence of stacking.
  • Stacked chromophores can have absorption spectra with (i) a characteristic decrease in the principal absorption peak as chromophore concentration increases and (ii) a characteristic shoulder peak ('band splitting').
  • rhodamine chromophores can form dimers at high concentrations in solution.
  • the dimer has a different absorbance spectrum from the monomer, and has little or no fluorescence in comparison with the monomer.
  • Two rhodamine chromophores attached to suitable positions in the protein can form dimers, whose interaction is altered when ligand binds to the protein.
  • the invention can spectroscopically detect the difference between the ATP-free and ATP-bound conformations of ATP binding molecule.
  • Molecular stacking takes place through the physical interaction of ground states of the two moieties. Labels that can undergo molecular stacking are well known in the art. Stacking can occur between identical chromophores, and can also occur between different chromophores.
  • the reporter moiety is a dye.
  • the reporter moiety is or comprises a fluorophore.
  • said fluorophore is attached at a position on the polypeptide such that conformational change of the polypeptide upon ATP binding causes a corresponding change in fluorescence of said fluorophore.
  • ATP sensor molecules with single labelling. Most suitably, when the ATP sensor is singly labelled a coumarin type dye is used as the label.
  • Double labelling means two dye molecules per sensor molecule. Suitably each of the two dye molecules is attached to a separate amino acid residue on the sensor molecule.
  • the dyes are rhodamine type dyes. For dual labelling, any stacking rhodamine type dye is useful.
  • Cy dyes may be useful in the invention. However, these might require different attachment points from those taught for rhodamine attachment due to different dye molecule sizes. In other words, the dye molecules may need to be placed closer together or further apart in space than the corresponding rhodamines. In some embodiments, suitably Cy dyes are not used, the reason is that Cy dyes can suffer from the drawback of tending to provide high fluorescence for much of the time. This can make it more difficult to observe the fluorescence changes which are useful in the invention.
  • Acrylodan dyes may be useful in the invention.
  • any rhodamine dye that can be specifically linked to surface thiols could be used. This includes the 5- and 6-isomers of tetramethylrhodamine.
  • Rhodamine dyes are available in different isomers.
  • 5- tetramethylrhodamine (5-ATR) and/or 6-ATR may be used to label ATP sensors of the invention.
  • 5-ATR and 6-ATR dye is used to label the sensor protein, the following species will be generated -
  • the sensor of the invention may comprise any of these species of molecules.
  • the sensor of the invention may comprise a mixture of more than one of these sensor molecules.
  • the sensor of the invention may comprise a mixture of all four of these labelled sensor molecules.
  • any suitable dye may be used, most suitably a dye which provides a coplanar alignment when attached to the sensor protein.
  • Other conformations are possible, for example a twisted conformation, but those often tend to provide a lower fluorescence.
  • a lower fluorescence may be usable but would need to be checked on a case by case basis.
  • any coumarin type dye is expected to provide good results.
  • any fluorophore known to have an environmentally sensitive fluorescence may be useful in the invention.
  • any coumarin may be used, including the iodoacetamide- and maleimide- linked diethylaminocoumarins (N-[2-(i-maleimidyl)ethyl]-7-diethylaminocoumarin-3- carboxamide and N-[2-(iodoacetamido)ethyl]-7-diethylaminocoumarin-3- carboxamide).
  • fluorophore types known to have fluorescence intensity, depending on physical environment , such as interactions with protein surfaces, include
  • MIANS (2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid)
  • IAEDANS (5-[2-[(2-Iodo-i-oxoethyl)amino]ethylamino]-i-naphthalenesulfonic acid) Alexa Fluor 488 maleimide
  • Cy3-maleimide (i-(6- ⁇ [2-(2,5-dioxo-2,5-dihydro-iH-pyrrol-i-yl)ethyl]amino ⁇ -6- oxohexyl)-2-[(iE,3E)-3-(i-ethyl-3,3-dimethyl-5-sulfo-i,3-dihydro-2H-indol-2- ylidene)prop-i-enyl]-3,3-dimethyl-3H-indolium)
  • Reporter moieties or labels such as fluorophores may be attached to the ATP binding molecule of the invention by any suitable means known in the art. Suitable amino acid residues may be engineered into the polypeptide.
  • cysteines such as cysteine substitutions
  • reporter e.g. fluorophores
  • alternate technologies such as peptide ligation (e.g. chemical synthesis of protein or attachment of synthetic peptides to other polypeptides), and/or introduction of unnatural amino acids using mutated tRNAs and/or tRNA synthetases may be used.
  • amino acids having reactive azide groups may be introduced to take advantage of 'click chemistry' (or vice versa) or other conjugation techniques may be used.
  • lysine based unnatural amino acids may be introduced to achieve this goal (e.g. Nguyen et al 2009 (J Am Chem Soc. 2009 Jul i;i3i(25):8720-i); e.g. Lang et al 2012 Nature Chemistry 4, 298-304 (2012)).
  • the fluorophores are attached by conventional conjugation techniques such as covalent attachment via a cysteine residue in the polypeptide component of the ATP binding molecule.
  • extrinsic reporter moieties to proteins are well known. Different cysteine residues show different reactivities to labelling reagents, which can be assessed using DTNB (5,5'-dithio-bis(2-nitrobenzoic acid)). Reporter moieties can be attached via amines or carboxyl residues on amino acid side chains, but it is more suitable to use covalent linkage via thiol groups on a cysteine residue. Where more than one label is attached to a protein, these are suitably attached to separate amino acid residues. Where a cysteine residue has to be introduced, either by insertion or substitution, a number of factors should be considered. These are discussed in more detail herein.
  • Exemplary sites for introduction of Cys residues and thus for label attachment are disclosed in detail herein. If attached chromophores are to interact, the residues must be selected such that they are in proximity to each other, and that the conformational change that occurs on ATP- binding affects one or both of the residues to cause a change in position or orientation or electronic environment of a label attached thereto. Exemplary pairs of attachment sites are set out above.
  • a key concept of the invention is that the ATP binding molecules (sensor proteins) are configured so that they undergo a conformational change upon ATP binding. It is detection of this conformational change which allows the ATP binding status of the molecules of the invention to be determined.
  • the ATP binding molecules sensor proteins
  • determination of the conformational status of the ATP binding molecule is suitably assessed as a population effect.
  • assessing the conformational change of an ATP binding molecule of the invention may be carried out by determining the conformational change of a population of ATP binding molecules of the invention.
  • the reporter moiety will be considered to be a fluorophore.
  • the fluorophore is attached to the ATP binding molecule of the invention.
  • ATP binding leads to a conformational change of the ATP binding molecule.
  • This conformational change can lead to a change in fluorescence.
  • This change in fluorescence may be an increase or a decrease upon ATP binding depending on the particular labelling strategy used. For any given application having a fixed amount of sensor protein, the change in fluorescence will be consistently associated with the corresponding change in ATP binding.
  • ATP binding is proportional to the concentration of ATP present in the sample being studied. Therefore, changes in ATP binding provide information about changes in the ATP concentration in the sample being studied. Thus, changes in fluorescence which are catalysed by conformational changes in the ATP binding molecule of the invention brought about by ATP binding directly provide information about the concentration of ATP in the sample being studied. For the great majority of applications or embodiments of the invention, conformational changes will be detected for a population of ATP binding molecules according to the invention. In practical terms, this means that a certain amount of the ATP binding molecule of the invention will be added to the sample being studied. The fluorescence of this population of ATP binding molecules of the invention will then be monitored.
  • a standard curve may be constructed by measuring the fluorescence of a constant amount of the ATP binding molecule of the invention in the presence of differing known concentrations of ATP. This standard curve may then be used in order to read out or convert measured fluorescence values to absolute concentrations of ATP present in a sample.
  • the readout of the invention may be advantageously calibrated by inclusion of samples having known ATP concentrations in the analysis being undertaken.
  • the samples containing known concentrations of ATP may be regarded as "internal controls". This permits accurate estimation of ATP concentrations in experimental settings where reference to a standard curve is less appropriate, for example in complex reaction mixtures in which other components might perturb the readouts, or might not have been present during the construction of a standard curve, thereby making such comparisons potentially inappropriate.
  • some of the sensors provided herein show increasing fluorescence in the presence of ATP, and some show decreasing fluorescence in the presence of ATP. Either type of sensor is useful.
  • sensors showing increased fluorescence upon binding ATP are used. These provide the advantage of avoiding confounding factors which might otherwise reduce fluorescence, for example photo-bleaching or other degradation of the dye. Occasionally a "percentage change" is discussed in the context of the invention.
  • the percentage change is the percentage increase in fluorescence from the unbound to the bound state.
  • the percentage change is calculated upwards i.e. taking the decreased fluorescence observed on binding and comparing that to the higher level of fluorescence observed in the absence of ATP gives a percentage increase.
  • a sensor whose fluorescence decreases from 1.0 to 0.6 upon ATP binding has a percentage fluorescence change of 67% (i.e. the difference in fluorescence intensity of 0.4 divided by the fluorescence on ATP binding of 0.6 equals 67% change).
  • Suitably sensors of the invention exhibit at least 50% fluorescence change upon ATP binding, more suitably 60%, more suitably 70%, more suitably 80%, more suitably 90%, more suitably 100% or even more (such as a multiple of fluorescence in the unbound state).
  • An ATP binding molecule is a molecule capable of binding ATP. Use of the term ATP binding molecule does not imply or require that ATP is present. ATP binding molecule means molecule capable of binding ATP.
  • Rhodopseudomonas palustris malonyl-coenzymeA synthetase (RpMatB) - Protein Data Bank (PDB) - 5-iodoacetamidotetramethylrhodamine (5-IATR) - 7-diethylamino-3- ((((2-maleimidyl)ethyl)amino)carbonyl)coumarin (MDCC) - 7-diethylamino-3-(((2- iodoacetomido)ethyl)amino)carbonyl)coumarin (IDCC)) - size-exclusion chromatography coupled to multi-angle laser light scattering (SEC-MALLS) - nicotinamide adenine dinucleotide (NADH) - deoxyadenosine triphosphate (dATP) - adenosine 5'(Y-thio)triphosphate (ATPyS) - aden
  • the senor of the invention is faster than any existing ATP sensor molecule such as those based on polypeptides.
  • An existing ATP sensor molecule known as "ATeam” has a 30 fold higher affinity for ATP than for ADP.
  • the sensors of the invention advantageously have approximately 67 fold higher affinity for ATP than for ADP. This is a significant advantage offered by sensors of the invention.
  • the exemplary biosensors shown have the advantage of larger signal.
  • the ATP binding molecule of the invention is, and may be used as, a reagentless biosensor.
  • the invention advantageously provides a reagentless biosensor for ATP.
  • the invention advantageously provides a fluorescent reagentless biosensor for ATP.
  • Divalent cation such as divalent metal ion is required for ATP binding to the sensors of the invention.
  • Cd 2+ Cadmium
  • Mn 2+ manganese
  • Mg 2+ Mg 2+
  • MgCl 2 magnesium chloride
  • any other acceptable salt of the above mentioned divalent metal ions may equally be used provided it does not compromise the action of the assay. This is easily tested as set out below in the example section whilst varying the divalent cation (i.e. the salt) which is incorporated into the assay.
  • the invention relates to a kit comprising an ATP sensor molecule as described above together with a source of divalent Magnesium ion (Mg 2+ ).
  • Mg 2+ divalent Magnesium ion
  • the source of Mg 2+ is Magnesium Chloride.
  • ATP will be present as MgATP.
  • Mg2+ is present in excess over ATP in the assay of the invention.
  • Mg2+ is present at imM or more in the assay of the invention.
  • ATP is present as MgATP in the assay of the invention. pH
  • the sensors of the invention have the advantage of being usable under a wide range of pH conditions.
  • the pH of the assay is in the range 6.0 to 9.0. More suitably the pH of the assay is in the range 7.0 to 7.5.
  • the sensor molecules do not react to nucleotides other than ATP, nor to ATP analogues. In other words, it does not matter if these chemical entities are also present in the assay mixture, the sensor has the advantage of only reacting to ATP and therefore the presence of these other molecular species does not perturb the assay of ATP concentration according to the invention.
  • the invention provides a fluorescent, reagentless biosensor for ATP, suitably based on or derived from malonyl-coenzyme A synthetase.
  • Table C shows the characteristics bestowed on the sensor by making of the mutations as shown. Therefore, depending on the application(s) of the biosensor intended by the skilled worker, mutations from Table C should be chosen accordingly, or not made, as desired.
  • 'Parent' means exemplary Rho-MatB; however, the mutations in Table C are equally applicable to any other biosensor disclosed herein without requiring the specific set of mutations of Rho-MatB - these additional mutations of Table C are merely exemplified in the Rho-MatB background to illustrate the advantages and help understand the effects of the mutations which are described individually and may be applied to biosensors of the invention individually or in combination.
  • mutations disclosed in Table C are applied individually.
  • the invention provides a biosensor as described above further comprising one further mutation selected from those disclosed in Table C.
  • Mutating T167 provides the advantage of increased Kd for ATP whilst retaining excellent fluorescence ratio (F+/F-). This has the further technical benefit of widening the range of ATP concentrations that can be measured by the biosensor.
  • T167 is mutated to a small amino acid.
  • T167 is mutated to Alanine (A), Serine (S) or Glycine (G).
  • Glycine can make the chain flexible which may or may not be desired.
  • T167 is mutated to Alanine (A) or Serine (S), avoiding flexibility effect(s) of Glycine.
  • said polypeptide comprises an amino acid other than T at the position corresponding to 167 of SEQ ID NO: 1.
  • said polypeptide comprises Alanine (A), Serine (S) or Glycine (G) at the position corresponding to 167 of SEQ ID NO: 1. More suitably said polypeptide comprises Alanine (A) or Serine (S) at the position corresponding to 167 of SEQ ID NO: 1.
  • said polypeptide comprises Serine (S) at the position corresponding to 167 of SEQ ID NO: 1, which has the advantage of enhanced fluorescence ratio and enhanced Kd compared to Rho- MatB.
  • said polypeptide comprises Alanine (A) at the position corresponding to 167 of SEQ ID NO: 1, which has the advantage of greatly enhanced Kd whilst retaining the same advantageous fluorescence ratio as Rho-MatB.
  • Mutating S170 provides the advantage of increased fluorescence ratio (F+/F-) whilst retaining excellent Kd for ATP. This has the further technical benefit of a decreased ADP affinity, thereby increasing selectivity of ATP over ADP to >200 fold. This is especially advantageous in measuring ATP when ADP is also present.
  • S170 is mutated to a small amino acid.
  • S170 is mutated to Alanine (A) or Glycine (G).
  • Glycine can make the chain flexible which may or may not be desired.
  • S170 is mutated to Alanine (A), avoiding flexibility effect(s) of Glycine.
  • said polypeptide comprises an amino acid other than S at the position corresponding to 170 of SEQ ID NO: 1.
  • said polypeptide comprises Alanine (A), or Glycine (G) at the position corresponding to 170 of SEQ ID NO: 1.
  • said polypeptide comprises Alanine (A) at the position corresponding to 170 of SEQ ID NO: 1.
  • said polypeptide comprises Alanine (A) at the position corresponding to 170 of SEQ ID NO: 1, which has the advantage of greatly increased fluorescence ratio (F+/F-) compared to Rho-MatB.
  • Mutating T303 provides the advantage of increased Kd for ATP whilst retaining excellent fluorescence ratio (F+/F-). This has the further technical benefit of widening the range of ATP concentrations that can be measured by the biosensor.
  • T303 is mutated to a small amino acid.
  • T303 is mutated to Alanine (A), Serine (S) or Glycine (G).
  • Glycine can make the chain flexible which may or may not be desired.
  • T303 is mutated to Alanine (A) or Serine (S), avoiding flexibility effect(s) of Glycine.
  • said polypeptide comprises an amino acid other than T at the position corresponding to 303 of SEQ ID NO: 1.
  • said polypeptide comprises Alanine (A), Serine (S) or Glycine (G) at the position corresponding to 303 of SEQ ID NO: 1. More suitably said polypeptide comprises Alanine (A) or Serine (S) at the position corresponding to 303 of SEQ ID NO: 1.
  • polypeptide comprises Serine (S) at the position corresponding to 303 of SEQ ID NO: 1, which has the advantage of enhanced Kd compared to Rho-MatB.
  • polypeptide comprises Alanine (A) at the position corresponding to 303 of SEQ ID NO: 1, which has the advantage of enhanced Kd whilst also retaining an advantageous high fluorescence ratio comparable to that of Rho-MatB.
  • positions corresponding to T167 and T303 of SEQ ID NO: 1 are not both mutated in the same sensor molecule.
  • double mutation of positions 167 and 303 may weaken ATP binding in such a double mutant.
  • the position corresponding to T303 is wild type, suitably T.
  • the position corresponding to T167 is wild type, suitably T.
  • the additional mutations described are made in conjunction with the mutations of RhoMatB such as shown in SEQ ID NO: 6.
  • the dyes and any other additions to the sensor molecule are as for RhoMatB.
  • the invention finds use as a reagent in a research setting.
  • the invention finds application in the assessment of food contamination, such as bacterial contamination.
  • the invention finds application in drug screening methodology.
  • the invention finds application in any setting in which it is desired to assay ATP.
  • the invention finds application in the assessment of surface contamination such as in a clinical/hospital/medical environment.
  • the invention finds application in the assessment of surface contamination such as in food preparation areas.
  • Figure 1 C-terminal domain rotation upon MgATP binding and position of mutations in RpMatB.
  • A Shown is a cartoon representation of RpMatB.
  • the C-terminal domain (amino acids 400-503) in the apo conformation is shown in pink.
  • the C-terminal domain in the MgATP-bound conformation (PDB 4FUT (Crosby, Rank et al.
  • Figure 2 Characterization of the fluorescence and absorbance spectra of Rho- MatB.
  • A Fluorescence excitation and emission spectra of 1 ⁇ Rho-MatB in 50 mM Hepes pH 7.0, 100 mM NaCl, 10 mM MgCl 2 and 0.3 mg ml "1 bovine serum albumin in the absence and the presence of 175 ⁇ ATP. Excitation was at 553 nm for the emission spectra. Emission was measured at 575 for the excitation spectra. Data were corrected for dilution.
  • Rho-MatB Titration of ATP (open circles), ADP (solid circles), dATP (open triangles), ATPyS (solid triangles) and AMP-PNP (open squares) to 0.5 ⁇ Rho-MatB in 50 mM Hepes pH 7.0, 100 mM NaCl, 10 mM MgCl 2 , 0.3 mg ml "1 bovine serum albumin. Aliquots of ligand were added and the fluorescence intensity was measured at 571 nm (exciting at 553 nm) at 20 °C. The data were corrected for dilution and normalized to 1 for the fluorescence intensity of Rho-MatB in the absence of ligand.
  • the dissociation constants were obtained using a quadratic binding curve (see Materials and methods). Shown here is one representative experiment. The dissociation constants listed in Table 1 are the average of at least three repeat experiments.
  • the fluorescence intensity was measured at 571 nm (exciting at 553 nm) at 20 °C.
  • the data were normalized to 1 for the fluorescence intensity of Rho-MatB in the absence of ATP.
  • the fluorescence response could be fitted to a line up to 6 ⁇ ATP with - for the experiment shown - slopes of 0.178 ⁇ 0.010, 0.180 ⁇ 0.004, 0.153 ⁇ 0.005 and 0.124 ⁇ 0.004 ⁇ "1 at 0, 10, 50 and 100 ⁇ ADP, respectively.
  • the long time scale fluorescence signal fitted to a double exponential function using a fixed rate constant for the fast phase determined previously from the short time scale traces.
  • the average rate constant determined for the slow phase is 0.88 ⁇ 0.13 s "1 .
  • the observed rate constants for the fast phase (k 0 b S ,fast) are plotted against ATP concentration (C). Linear regression gave an association rate constant of 1.83 ⁇ 0.02 ⁇ "1 s "1 and the intercept was 8.15 ⁇ 0.17 s "1 .
  • Rho-MatB Association kinetics were measured under pseudo-first order conditions with a 5 or 10-fold excess of Rho-MatB over ATP (0.25 ⁇ ATP and 1.25 ⁇ Rho-MatB; 0.50 ⁇ ATP and 2.50 ⁇ Rho-MatB; 0.375 ⁇ ATP and 3.75 ⁇ Rho-MatB; 0.500 ⁇ ATP and 5.00 ⁇ Rho-MatB; 0.625 ⁇ ATP and 6.25 ⁇ Rho-MatB; 0.750 ⁇ ATP and 7.50 ⁇ Rho-MatB or 1.00 ⁇ ATP and 10.0 ⁇ Rho-MatB).
  • Rho-MatB2 can bind ATP to form ATP RhoMatB2.
  • Rho-MatB binds ATP to form ATP RhoMatB.
  • ATP RhoMatB* corresponds to ATP-bound Rho-MatB in a different conformational state.
  • Figure 6 The production of ATP by pyruvate kinase as monitored by Rho-MatB.
  • the initial rates (vi) were determined by linear regression (from 200 to 400 s) using the slope obtained from the calibration curve and were plotted versus phosphoenolpyruvate concentration (C).
  • the parameters K m and Vmax were obtained from a curve fit according to the Michaelis-Menten equation and are - for the experiment shown - 75.5 ⁇ 3.4 ⁇ and 0.019 ⁇ 0.002 ⁇ s "1 , respectively.
  • D Time courses of ATP production by pyruvate kinase as monitored by Rho-MatB in a stopped-flow apparatus.
  • Reaction mixtures contained 50 mM Tris HC1 pH 7.5, 100 mM NaCl, 10 mM MgCl 2 , 100 mM KC1, 0.3 mg ml "1 bovine serum albumin, 250 ⁇ ADP, 100 ⁇ phosphoenolpyruvate and 2.5 ⁇ Rho-MatB. All reactions were started by the addition of pyruvate kinase (0.025, 0.50, 0.75, 1.0, 2.0, 4.0, 5.0 or 6.0 U ml "1 ), and the change in fluorescence was monitored for several seconds at 25 °C. ATP concentrations were calculated from the fluorescence signal using the calibration method.
  • the signal was calibrated by consecutively introducing 2.5 ⁇ Rho-MatB alone in 50 mM Tris HC1 pH 7.5, 100 mM NaCl, 10 mM MgCl 2 , 100 mM KC1, 0.3 mg ml "1 bovine serum albumin, 250 ⁇ ADP, 100 ⁇ phosphoenolpyruvate; then the same solution containing 0.5, 1.0, 2.0, 3.0, 4.0 or 5.0 ⁇ ATP.
  • the fluorescence was measured.
  • a representative set of traces at different pyruvate kinase concentrations is shown. Traces are offset by 0.20 ⁇ ATP from each other at zero time for clarity.
  • the initial rates (vi) were determined by linear regression and were plotted versus pyruvate kinase concentration (E).
  • Figure 7 Chromatogram of the purification of Rho-MatB via ion exchange chromatography. Detection was performed via A 28 onm- The conductivity (red) is shown on the secondary vertical axis. The conductivity signal gives an indication of the applied gradient. See Materials and methods for further details.
  • Figure 8 (Supplemental figure 2): Characterization of the fluorescence spectra of Rho-MatB.
  • A Fluorescence emission spectra of 1 ⁇ Rho-MatB in 50 mM Hepes pH 7.0, 100 mM NaCl, 10 mM MgCl 2 and 0.3 mg ml "1 bovine serum albumin (black line) supplemented with 1 mM malonate (red line), 0.25 mM coenzymeA (blue line), 1 mM malonate and 0.25 mM coenzymeA (green line) or 1 mM malonate, 0.25 mM coenzymeA and 1 mM ATP (orange line). Data were corrected for dilution.
  • Simulation using the conformational selection model (Figure 5A). Simulated time traces shown are, from bottom to top, 0.25 ⁇ ATP : 1.25 ⁇ Rho-MatB; 0.375 ⁇ ATP: 3.75 ⁇ Rho-MatB; 0.625 ⁇ ATP: 6.25 uM Rho-MatB and 1.00 ⁇ ATP: 10.0 ⁇ Rho-MatB.
  • the simulated time traces are normalized to 100% for the initial signal but offset by 2% from each other for clarity.
  • Rho- MatB 1 and Rho-MatB2 concentrations were both set at 50 % of the total [Rho-MatB].
  • Simulated time traces shown are, from bottom to top, 0.25 ⁇ ATP : 1.25 ⁇ Rho-MatB; 0.375 uM ATP: 3.75 ⁇ Rho-MatB; 0.625 ⁇ ATP: 6.25 ⁇ Rho-MatB and 1.00 ⁇ ATP: 10.0 ⁇ Rho- MatB.
  • the simulated time traces are normalized to 100% for the initial signal but offset by 2% from each other for clarity.
  • Figure 10 (Supplemental figure 4): Rho-MatB activity assay.
  • Reaction mixtures contained 50 mM Hepes buffer pH 7.5, 25 mM NaCl, 25 mM KC1, 0.3 mg ml "1 bovine serum albumin, 0.5 mM ATP, 0.5 mM coenzymeA, 10 mM MgCl 2 , 3 mM phosphoenolpyruvate, 0.2 mM NADH, 0.01 U ⁇ "1 pyruvate kinase, 0.05 U ⁇ "1 adenylate kinase, 0.015 U ⁇ "1 lactate dehydrogenase and 2 mM malonate.
  • the reaction was started by the addition of 0.03 ⁇ Rho-MatB and the change in A34 0n m was monitored in time. No activity was observed.
  • 0.03 ⁇ (5-ATR) 2 -His 6 -RpMatB C106A/R286C/Q457C was added to the reaction mixture.
  • the specific activity for (5- ATR) 2 -His 6 -RpMatB C106A/R286C/Q457C was calculated using 8 3 40nm,NADH and was 37 ⁇ 4 ⁇ AMP min "1 mg "1 .
  • Figure 11 (Supplemental figure 5): The production of ATP by pyruvate kinase as monitored by a coupled-enzyme assay.
  • Reaction mixtures 200 ⁇ contained 50 mM Tris HCl pH 7.5, 100 mM NaCl, 10 mM MgCl 2 , 100 mM KC1, 0.3 mg ml "1 bovine serum albumin, 250 ⁇ ADP, 200 ⁇ NADH, 5 U ml "1 lactate dyhydrogenase and various concentrations of phosphoenolpyruvate.
  • Rho-MatB blue
  • His 6 -RpMatB C106A/R286C/Q457C/K488A were analyzed via SEC -MALLS at room temperature at a flow rate of 0.5 ml min "1 in the absence of ATP (30 mM Tris HCl pH 7.5, 100 mM NaCl, 3 mM NaN 3 , graph on the left) and in the presence of ATP and Mg 2+ (30 mM Tris HCl pH 7.5, 100 mM NaCl, 5 mM MgCl 2 , 50 ⁇ ATP, 3 mM NaN 3 , graph on the right).
  • RhoMatB in the absence of ATP or Mg 2+ , we observe a broad peak with fronting corresponding to a molar mass ranging from -60 to 80 kDa for His 6 -RpMatB C106A/R286C/Q457C/K488A and a molar mass ranging from -60 to 90 kDa for RhoMatB.
  • the equilibrium shifts toward the 60 kDa form (data not shown).
  • Addition of 50 ⁇ ATP in the presence of 5 mM Mg 2+ shifts the equilibrium towards the 60 kDa species for the unlabeled protein.
  • Rho-MatB i.e. its molar mass ranges from -60 to 80 kDa.
  • Figure 13 (Table 1): Fluorescence changes (F + /F.) and dissociation constants (K d ) for binding of ATP and other ligands to Rho-MatB. The fluorescence change upon ligand binding and the equilibrium dissociation constants were obtained from fluorescence titrations as described in Figure 3A and Figure 3B.
  • Figure 14 (Supplemental table 1): Fluorescence changes (F + /F.) and dissociation constants (K d ) for binding of ATP and ADP to diethylaminocoumarin (MDCC and IDCC) and tetramethylrhodamine (5-ATR and 6-ATR) labeled RpMatB cysteine mutants. Data are from a survey without complete optimization of the labeling and purification for each mutant.
  • the fluorescence changes upon ligand binding were obtained from fluorescence emission spectra at 20 °C in 50 mM Tris HC1 pH 7.5, 100 mM NaCl, 10 mM MgCl 2 , 0.3 mg / ml bovine serum albumin using 1 ⁇ protein and excess ATP or ADP.
  • the equilibrium dissociation constants were obtained from fluorescence titrations at 20 °C in 50 mM Tris HC1 pH 7.5, 100 mM NaCl, 10 mM MgCl 2 , 0.3 mg / ml bovine serum albumin using 0.5 ⁇ protein and various concentrations of ATP or ADP.
  • Figure 15 Fluorescence changes (F + /F.) and dissociation constants (K d ) for binding of ATP to Rho-MatB in different buffer conditions.
  • the fluorescence changes upon ATP binding were obtained from fluorescence emission spectra at 20 °C in the buffer mentioned using 1 ⁇ protein and excess ATP in the presence of 10 mM MgCl 2 and 0.3 mg ml "1 bovine serum albumin.
  • the equilibrium dissociation constants were obtained from fluorescence titrations at 20 °C in the buffer mentioned using 0.5 ⁇ protein and various concentrations of ATP.
  • Figure 16 (Supplemental Table 3): Structure comparison between apo and holo (ATP (analogue) -bound) ANL superfamily proteins.
  • Figure 17 shows an exemplary sequence of the invention referred to as RpMatB, which is annotated to show examples of substitutions and/ or additions useful in sensors of the invention (SEQ ID NO: 7).
  • FIG. 18 shows summary of the invention.
  • Figure 19 shows structural comparison of the proteins in Table B. Location of amino acids closest to those used in RpMatB as cysteine points of attachment.
  • Figure 20 shows C-terminal domain rotation upon MgATP binding and position of mutations in RpMatB.
  • Figure 21 shows Crystal structure of RpMatB in the MgATP bound conform ation, showing conserved m otifs and position of binding site m utations.
  • Figure 22 shows Fluorescence excitation and em ission spectra of variants of Rho-MatB.
  • A l ⁇ Rho-MatB T167A with and without 5 niM ATP;
  • B 1 ⁇ Rho-MatB T303A with and without 3 mM ATP;
  • C 1 ⁇ Rho-MatB S170A with and without 0.5 mM ATP.
  • ATP concentrations were saturating for the variant. Solutions were in 50 mM Hepes pH 7.0, 100 mM NaCl, 10 mM MgCl 2 , 0.3 mg ml 1 bovine serum albumin at 20 °C.
  • Figure 23 shows Nucleotide affinity to variants of Rho-MatB.
  • Calibrations were determined by measuring the fluorescence as a function of ATP with different amounts of ADP present, but with the total nucleotide concentration (ADP + ATP) constant.
  • A 1 ⁇ Rho-MatB T167A
  • B 1 ⁇ Rho-MatB T303A
  • C 1 ⁇ Rho- MatB S170A.
  • the total nucleotide concentration is shown in micromolar.
  • 500 ⁇ ADP was added at each ATP concentration. Solution conditions were as in Figure 23. The data were linear fit to demonstrate approximate linear dependence over the range measured.
  • Figure 25 shows Association kinetics of ATP binding to Rho-MatB variants Fluorescence time courses were measured by rapidly mixing different concentrations of ATP with 0.25 ⁇ Rho-MatB with a large excess of ATP (micromolar concentration shown) in 50 mM Hepes pH 7.0, 100 mM NaCl, 10 mM MgCl 2 , 0.3 mg ml 1 bovine serum albumin at 20 °C. Time courses are for two different time scales were obtained to show fast and slow phases. Slow phases are shown in Figure 28 ( Figure S3). Note that the dead time of the stopped-flow instrument is ⁇ 2 ms, so that the traces of the fast phase only record changes from that time.
  • Figure 26 shows core m otifs sequences Sequence logos were created for ANL superfamily proteins, using WebLogo 3.4 Sequence conservation is indicated as the total height of each stack (measured in bits), while the relative height of bases in a stack reflects base frequencies at that position. The numbers correspond to the alignment position.
  • the colour scheme is based on hydrophobicity: R, K, D, E, N, Q are blue; S, G, H, T, A, P are green; Y, V, M, C, L, F, I, W are black.
  • the motifs shown are ones in which mutations were prepared: the sequence of RpMatB is also shown for each.
  • Figure 27 shows Absorbance spectra of variants of Rho -MatB with and without ATP
  • A 1 ⁇ Rho-MatB T167A with and without 5 mM ATP
  • B 1 ⁇ Rho-MatB T303A with and without 3 mM ATP
  • C 1 ⁇ Rho-MatB S170A with and without 0.5 mM ATP.
  • ATP concentrations were saturating for the variant. Solutions were in 50 mM Hepes pH 7.0, 100 mM NaCl, 10 mM MgCl 2 , 0.3 mg ml 1 bovine serum albumin at 20 °C.
  • Figure 28 shows Association kinetics of variants of Rho -MatB with excess ATP
  • Example time courses were obtained as in Figure 25 at various ATP concentrations, shown in micromolar.
  • Figure 25 shows the fast phases of each time course, the equivalent slow phase are shown here. These were fit to single exponentials, whose rate constants varied little with ATP concentration. The average rate constants, measuring a conformation change as described in the main text, are in Table D.
  • Plasmid pRpMatE>39 (plasmid pTEVs containing the coding sequence of RpMatB with the point mutation K488A and an N-terminal His 6 -tag) was provided by J.C. Escalante- Semerena (Co2. ⁇ ...EanJ ...et...a]....20i2).
  • the QuikChange site-directed mutagenesis protocol and the QuikChange Lightning Multi site-directed mutagenesis kit (Stratagene) were used for single-site or multi-site mutations of the pRpMatE>39 plasmid, respectively.
  • the primers to introduce the point mutation C106A in the pRpMatE>39 plasmid are 5'-ccgaagatcgtggtggccgatccgtccaagcg-3' and 5'- cgcttggacggatcggccaccacgatcttcgg-3'.
  • the primers to introduce R286C and Q457C, i.e. the mutations in the most preferred RpMatB biosensor are respectively 5'- gctcgccgatacgcattgcgaatggtcg-3' and 5'-acgatcgacgaagcgtgcgtgcacggcctc-3'.
  • RpMatB variants were synthesized in E. coli OverExpress C4i(DE3) cells (Lucigen).
  • the cultures were cooled down to 4 °C and centrifuged at 3500 rpm for 30 min at 4 °C (rotor JS 4.2, Beckman).
  • the cell pellet was washed with 30 ml of ice-cold buffer (10 mM Tris HCl pH 7.5, 300 mM NaCl), centrifuged at 3500 rpm for 30 min at 4 °C (rotor JS 4.2, Beckman), the supernatant discarded and the pellet stored at -80 °C until use.
  • About 3 g wet weight of E. coli cells were harvested from 0.5 1 culture in a typical preparation.
  • the cell pellet was resuspended in 35 ml 30 mM Tris HCl, 300 mM NaCl, 10 mM imidazole, 3 mM tris(2-carboxyethyl)phosphine, 2 mM phenylmethanesulfonyl fluoride, pH 8.0 and sonicated on ice using an ultra-sonicator (VC505, Sonics) at 200 W for 5 times 30 s with a 5 s on / 5 s off pulser.
  • the soluble fraction was collected by centrifugation at 35000 rpm for 45 min at 4 °C (rotor 45 Ti, Beckman).
  • the His 6 -tagged protein was purified at 4 °C on an immobilized metal ion affinity chromatography (1 ml HisTrap HP column, GE Healthcare) using an Akta system (GE Healthcare).
  • the resin was equilibrated with Buffer A (30 mM Tris HCl, 300 mM NaCl, 10 mM imidazole, 1 mM tris(2-carboxyethyl)phosphine, pH 8.0).
  • Buffer A (30 mM Tris HCl, 300 mM NaCl, 10 mM imidazole, 1 mM tris(2-carboxyethyl)phosphine, pH 8.0).
  • the sample was filtered (0.45 ⁇ Minisart NML filter, Sartorius) and loaded onto the column at 0.5 ml min 1 .
  • the column was washed with 20 ml Buffer A and additionally with 20 ml of 95 % Buffer A and 5 % Buffer B (30 mM Tris HCl, 300 mM NaCl, 250 mM imidazole, 1 mM tris(2- carboxyethyl)phosphine, pH 8.0) at a flow rate of 1 ml min 1 .
  • the protein was eluted with 20 ml of Buffer B at a flow rate of 1 ml min 1 .
  • Protein fractions were pooled ( ⁇ 2-4 ml) and further purified via size exclusion chromatography at 4 °C using the HiLoad 16/60 Superdex 200 prep grade column (GE Healthcare) equilibrated with 30 mM Tris HCl, 100 mM NaCl, 0.5 mM ethylenediaminetetraacetic acid, 5 mM dithiothreitol, 1 mM NaN 3 . The flow rate was 1 ml min 1 and the loading volume ranged from 2 to 4 ml. Fractions containing the protein were pooled and concentrated (VivaSpin 20 MWCO 10 kDa cut off, GE Healthcare) to ⁇ io mg ml 1 .
  • the protein concentration was determined from the absorbance at 280 nm using the extinction coefficient at 280 nm of 46300 M 1 cm 1 calculated from the sequence via Expasy Protparam (Wilkins . , Gasteiger et al. 1999).
  • the protein was drop-frozen in liquid nitrogen and stored at -80 °C. Typically, 45 mg of protein was obtained from 3 g wet weight of cells.
  • Dithiothreitol was removed from ⁇ 40 mg of protein using a PD10 desalting column (GE Healthcare) pre- equilibrated with Buffer L (30 mM Tris HCl pH 7.5, 100 mM NaCl) at 20 °C. 50 ⁇ protein was incubated at 20 °C with 225 ⁇ 5- iodoacetamidotetramethylrhodamine (5-IATR, AnaSpec, CA) in Buffer L using an end- over-end mixer for 90 min. Afterwards, 2 mM sodium-2-mercaptoethanesulfonate was added and incubation continued for 15 min.
  • Buffer L 30 mM Tris HCl pH 7.5, 100 mM NaCl
  • the most preferred RpMatB biosensor was further purified via ion exchange chromatography at 4 °C using a 1 ml HiTrap Q HP column (GE Healthcare), equilibrated in Buffer Qi. The flow rate was 1 ml min 1 during the whole purification. After sample loading, the column was washed with 90 ml Buffer Qi. The protein was eluted using a gradient from 100 % Buffer Qi to 50 % Buffer Qi and 50 % Buffer Q2 (30 mM Tris HC1 pH 8.0, l M NaCl) over 25 ml followed by a gradient from 50 % Buffer Qi and 50 % Buffer Q2 to 100 % Buffer Q2 over 10 ml. Fractions containing the protein were pooled and concentrated to ⁇ 5 mg ml 1 using a concentrator (Amicon Ultra-4 10 kDa cut off, Millipore).
  • RpMatB variants (100 ⁇ ) were incubated at 20 °C with 2-fold (7-diethylamino-3-((((2-maleimidyl)ethyl)amino)carbonyl)coumarin (MDCC) or 7-diethylamino-3-((((2-iodoacetomido)ethyl)amino)carbonyl)coumarin (IDCC)) or 4- fold (5-IATR or 6-IATR (synthesized in-house (C rno and Craik : Q )) ) excess of fluorophore over RpMatB for 90 (tetramethylrhodamine), 35 (MDCC) or 120 (IDCC) min.
  • MDCC 7-diethylamino-3-((((2-maleimidyl)ethyl)amino)carbonyl)coumarin
  • IDCC 7-diethylamino-3-((((2-iodoacetomid
  • the labeled protein concentrations were determined using the following extinction coefficients: RpMatB: 8 2 8onm (46300 M “1 cm 1 ), tetramethylrhodamine: 8 2 8onm (31000 M “ 1 cm “1 ) and 8 52 8nm (52000 M “1 cm “1 ) (Conic and Cr;;ik 0 4).
  • MDCC 8 2 8onm (7470 M “1 cm- and 8 43 onm (46800 M “1 cm “1 ) and IDCC: 8 2 8onm (7470 M “1 cm “1 ) and 8 43 onm (44800 M “1 cm 1 ).
  • the protein was drop-frozen in liquid nitrogen and stored at -80 °C. Labeling yields were up to 35 %.
  • RpMatB His 6 -RpMatB C106A/R286C/Q457C/K488A
  • the theoretical molecular weight is 57324.2 Da, assuming loss of the N-terminal methionine.
  • other post-translational modifications such as partial oxidation of surface-accessible methionines, occur (Gtian..Yate calls..e . : ..20.Q ).
  • Rho-MatB ((5-ATR) 2 -His 6 -RpMatB C106A/R286C/Q457C/K488A)
  • Rho-MatB ((5-ATR) 2 -His 6 -RpMatB C106A/R286C/Q457C/K488A)
  • the solution molecular weight was analyzed using size exclusion chromatography coupled to multi-angle laser light scattering (SEC-MALLS). Protein (1 mg ml 1 ) was applied in a volume of 100 ⁇ to a Superdex 200 10/300 GL column (GE Healthcare) connected to a Jasco PU-1580 HPLC at a flow rate of 0.5 ml min 1 .
  • the HPLC system was connected to a Dawn Heleos II light scattering instrument (Wyatt Technology) and Optilab T-rex differential refractometer (Wyatt Technology).
  • the solution molecular weight was determined from the combined data from both detectors using the ASTRA software version 6.1.1.17 (Wyatt Technology) with the refractive index increment set to 0.1860 ml g 1 .
  • RpMatB specific activity was quantified using a nicotinamide adenine dinucleotide (NADH) assay CCrosby ⁇ I ank et aL . 2012).
  • Reaction mixtures 200 ⁇ contained 50 mM Hepes buffer pH 7.5, 25 mM NaCl, 10 mM MgCl 2 , 25 mM KC1, 0.3 mg ml 1 bovine serum albumin, 0.5 mM ATP, 0.5 mM coenzymeA, 3 mM phosphoenolpyruvate, 0.2 mM NADH, 0.01 U ⁇ 1 pyruvate kinase (rabbit muscle (Sigma)), 0.05 U ⁇ 1 adenylate kinase (chicken muscle (Sigma)), 0.015 U ⁇ 1 lactate dehydrogenase (rabbit muscle (Sigma)) and 2 mM malonate. All reactions were started by the addition of RpMatB (0.03
  • Fluorescent measurements were obtained on a Cary Eclipse Spectrofluorometer (Agilent Technologies), using a 3-mm path-length quartz cuvette (Hellma), unless otherwise mentioned. Excitation and emission slits were set at 5 nm. Protein and nucleotide concentrations, buffer conditions and excitation and emission wavelengths used are given in the figure legends.
  • a calibration curve was determined using various concentrations of ATP added to the solution above in the presence of 2.5 mM phosphoenolpyruvate. Reactions were started by the addition of pyruvate kinase (0.025 U ml 1 ), and the change in fluorescence signal was monitored at 20 °C. Linear regression analysis was used to determine the initial velocity.
  • Stopped- flow Steady-state measurements of pyruvate kinase activity were obtained using a stopped-flow apparatus, as above.
  • the excitation wavelength was 548 nm and there was an OG570 cut-off filter on the emission.
  • Reaction mixtures contained 50 mM Tris HCl pH 7.5, 100 mM NaCl, 10 mM MgCl 2 , 100 mM KCl, 0.3 mg ml 1 bovine serum albumin, 250 ⁇ ADP, 100 ⁇ phosphoenolpyruvate and 2.5 ⁇ Rho-MatB.
  • a calibration curve was determined using various concentrations of ATP added to the solution above.
  • Finding a suitable candidate protein recognition element for an ATP biosensor requires comparing ATP- or ATP-analogue- bound protein structures with their corresponding ligand-free protein structures. If that comparison revealed a significant conformational change upon ligand binding, the protein was seen as a potential candidate for biosensor development. Such conformational changes can be harnessed to transduce ligand binding to a fluorescence change to a fluorophore reporter, local to that region of the protein so that it responds to the change in structural environment. Functional parameters were considered next, for example affinity and selectivity for ATP, or known mutants that block enzymatic activity, such as ATPase activity. Following this analysis, RpMatB was chosen as the most suitable candidate for further ATP-biosensor development.
  • Cysteine mutations were introduced as sites for labeling onto a background of the C106A and K488A mutations in the wild-type protein.
  • the K488A RpMatB variant does not catalyze the adenylation half-reaction, that is it cannot convert ATP and malonate to malonyl-AMP and pyrophosphate (Cro . sj3 5.. R k . ... aI ⁇ . 2Qi2).
  • C106 in the wild-type protein ( Figure lA) is situated in the N-terminal domain, distant from the active site but slightly solvent accessible. Having shown that there is a low, but significant, degree of background labeling at this position (6% with MDCC), C106 was mutated to alanine.
  • Sites for tetramethylrhodamine labeling were chosen so that stacking of the two fluorophores might be possible in the apo conformation and that dissociation of these stacked tetramethylrhodamines could occur on the conformational change with MgATP binding.
  • positions were chosen with a suitable distance (-1.5 nm) and orientation between them in the apo conformation.
  • the distance and orientation between the chosen positions changed when MgATP binds. Results from seven different combinations are in Supplemental Table 1.
  • Rho- MatB The labeled variant with the largest fluorescent increase upon MgATP-binding was (5- ATR) 2 -His 6 -RpMatB C106A/R286C/Q457C/K488A, hereinafter referred to as Rho- MatB. Both positions are well defined in the apo and MgATP-bound structure as shown in Figure lA. R286 is located in the N-terminal domain and Q457 in the C-terminal domain.
  • Rho-MatB The fluorescence of Rho-MatB responded to addition of nucleotides other than ATP. There was an increase in fluorescence intensity upon addition of ADP, deoxyadenosine triphosphate (dATP), adenosine 5'-(Y-thio)triphosphate (ATPyS) and adenosine 5'-( ⁇ , ⁇ - imido)triphosphate (AMP-PNP). In contrast, AMP, GDP and GTP did not have a significant effect. Also, RpMatB substrates other than ATP (i.e. malonate and coenzymeA) had no influence on the fluorescence and their presence did not inhibit the MgATP-induced fluorescence increase (Supplemental Figure 2A).
  • dATP deoxyadenosine triphosphate
  • ATPyS adenosine 5'-(Y-thio)triphosphate
  • AMP-PNP adenosine 5'-( ⁇ , ⁇ - imido)
  • the affinity for the responsive nucleotides was determined by measuring the fluorescence at different concentrations of the nucleotide in a solution of Rho-MatB ( Figure 3A and Table 1).
  • the dissociation constants for ADP, dATP, ATPyS and AMP-PNP were 428 ⁇ , 440 ⁇ , ⁇ 6.2 ⁇ and 253 ⁇ , respectively. So the binding of ADP, dATP and AMP-PNP to RpMatB was much weaker than ATP.
  • the affinity of ATPyS to Rho-MatB was similar to ATP. The maximum fluorescence increase upon binding these nucleotides was smaller than with ATP.
  • the fluorescence response to ATP was measured in different buffers, over a pH range from 6.0 to 9.0 and different ionic strengths with salt from 50 to 200 mM (Supplemental Table 2). Although the size of the fluorescence response changed with solution conditions, there were rather small effects on the 3 ⁇ 4 for ATP.
  • Mg 2+ is required for the fluorescence change, suggesting that MgATP is bound (Supplemental Figure 2B), as might be expected as Mg 2+ is a cofactor for the enzyme.
  • Exam ple 5 Binding kinetics
  • the kinetics of ATP binding and dissociation were measured by stopped-flow fluorescence in order to assess the range of rates over which Rho-MatB is suitable for real-time measurements.
  • Binding kinetics were first measured under pseudo-first-order conditions by rapidly mixing different concentrations of ATP, in large excess, with Rho-MatB (Figure 4A & 4B). The time course of the subsequent fluorescence response was biphasic, fitting well to a double exponential. The observed rate constant for the fast phase increased linearly up to 150 ⁇ ATP ( Figure 4C), giving a slope of 1.83 ⁇ 1 s 1 and the intercept was 8.2 s 1 . Assuming this phase represents binding, the slope is the association rate constant, the intercept is the dissociation rate constant. The dissociation constant, calculated from these values (4.5 ⁇ ) agrees well with the value from equilibrium binding data (Figure 3A and Table 1). The observed rate constants for the slow phase did not vary significantly over the ATP concentration range with an average value of 0.88 s 1 . The biochemical basis of these rate constants will be discussed later.
  • Dissociation kinetics were measured directly starting from Rho-MatB.ATP complex and trapping dissociated ATP with a large excess of unlabeled protein (Figure 4F).
  • the fluorescence decreased with time and the curves required a double exponential with rates 6.9 s 1 and 1.5 s 1 . These do not vary when the concentration of the unlabeled protein varies, indicating that the process is ATP-dependent.
  • Rho-MatB showed no residual activity under the conditions tested (Supplemental Figure 4).
  • Rho-MatB variant but without the K488 mutation ((5-ATR) 2 - His 6 -RpMatB C106A/R286C/Q457C) had a specific activity of 37 ⁇ 4 ⁇ AMP mg 1 min 1 .
  • Test assay Steady-state production of ATP by pyruvate kinase
  • the ATP biosensor was tested in a steady-state assay in which ATP was produced from ADP and phosphoenolpyruvate in a reaction catalyzed by pyruvate kinase ( Figure 6A and Figure 6B). This was chosen as there is a coupled-enzyme assay for this enzyme, that is well established and described below, which could be used to validate the biosensor results.
  • the rate of ATP formation was measured at different concentrations of phosphoenolpyruvate using Rho-MatB.
  • pyruvate formation was measured using a coupled-enzyme assay under the same conditions to compare the parameters directly.
  • This assay gave a K m of 251 ⁇ and a Vmax of 0.66 ⁇ s 1 U 1 ml 1 (Supplemental Figure 5).
  • the Vmax was similar to that obtained using Rho-MatB; reasons why the K m is different for the two assays probably relates to the different extents of reaction required to perform the two assays.
  • N-terminal His 6 -tag is highlighted in vzdf italics.
  • the mutations are highlighted in blue/ underlined and labeled.
  • the residues in close proximity (sequence and space) of the labeled positions are highlighted in gt*e *x/ bold. They are summarised in Table 2.
  • truncated forms are those which lack a small number of amino acid residues from the N- or C- terminus of the polypeptide relative to wild type MatB.
  • reasons for the range of observations may include:
  • introducing a fluorophore might have an effect on protein flexibility and therefore on the documented conformational change upon ligand binding (either because of interaction with amino acids or because of interactions between fluorophores).
  • Rho-MatB can be used as a biosensor for ATP under various pH and salt conditions (Supplemental Table 2) in the presence of Mg 2+ (Supplemental Figure 2B).
  • the maximum fluorescence increase upon ATP-binding was 3.7-fold ( Figure 2A).
  • the change in the absorbance spectrum provides support that this increase is due to the unstacking of the two tetramethylrhodamines upon ATP-binding ( Figure 2B).
  • Rho-MatB binds ATP with a dissociation constant of 6.4 ⁇ ( Figure 3A and Table 1), higher than RpMatB K488A (0.31 ⁇ ) CCm ⁇ : a ... nk .. et..d- ... 2Qi2).
  • HHoowweevveerr wwee ddoo hhaavvee ttoo ppooiinntt oouutt tthhaatt tthheerree iiss aa ddeevviiaattiioonn ffrroomm lliinneeaarriittyy 2200 ffoorr kk 00 bbss,,ffaasstt aatt hhiigghheerr AATTPP ccoonncceennttrraattiioonnss,, ssuuggggeessttiinngg ttwwoo--sstteepp bbiinnddiinngg..
  • HHoowweevveerr HHoowweevveerr, nnoo vvaalliidd rraattee ccoonnssttaannttss ccaann bbee oobbttaaiinneedd ffoorr AATTPP ccoonncceennttrraattiioonnss ooff mmoorree tthhaann 220000 ⁇ aatt 2255 °°CC dduuee ttoo tthhee ddeeaadd ttiimmee ooff tthhee iinnssttrruummeenntt..
  • TThheerreeffoorree nnoo aaccccuurraattee kkiinneettiicc ppaarraammeetteerrss ffoorr aa ttwwoo--sstteepp bbiinnddiinngg mmeecchhaanniissmm aarree aavvaaiillaabbllee..
  • Rho-MatB exists in two different conformations prior to ATP-binding. ATP binds to only one conformation. In excess Rho-MatB, there is “enough" Rho-MatB in the "correct” conformation to bind ATP and we observe only AATTPP--bbiinnddiinngg,, hheennccee ssiinnggllee pphhaassee bbiinnddiinngg kkiinneettiiccss..
  • tetramethylrhodamine has high photostability. Its fluorescence is unlikely to interfere with (or be affected by) the system being studied because of excitation around 550 nm end emission around 570 nm. Thus, this ATP biosensor has many advantages relative to other ATP assays.
  • Rho-MatB can be used as a sensitive probe to measure ATP. It shows a linear response in the micromolar range and is selective for ATP. It can be used to elucidate the mechanisms of ATP production.
  • This protein family contains acyl- and aryl-coenzymeA synthetases, the adenylation domains of nonribosomal peptide synthetases and firefly luciferases.
  • Structural information suggests a similar ligand-mediated conformational change for family members, even if the structures of ANL superfamily proteins lacking ligands are highly variable in the position of the C-terminus. This may be exploited to generate a family of ATP biosensors with possibly different properties (such as ATP sensitivity, fluorescence signal) using protein engineering techniques, based on straightforward structure and sequence principles.
  • the quality of the alignment will improve when performing a multiple sequence alignment instead of a pairwise alignment.
  • polypeptide component of the ATP sensor of the invention comprises sequence corresponding to one of the polypeptides in the above table.
  • Figure 20 shows cartoon representations of RpMatB.
  • the N-terminal domain (amino acids 1-399) in the apo conformation (PDB 4FUQ (Crosby, Rank et al. 2012)) is coloured grey.
  • the C-terminal domain (amino acids 400-503) in the apo conformation is shown in pink.
  • the C-terminal domain in the MgATP-bound conformation (PDB 4FUT (Crosby, Rank et al. 2012), after superimposing the N-terminal domain of 4FUT on the N-terminal domain of 4FUQ) is shown in green.
  • ATP in ball and stick conformation and coloured by CPK convention
  • Mg 2+ depicted as an orange sphere
  • a search for potentially related sequences of known structure can be performed by the profile.buildO command of MODELLER (manual page https://Milab.org/modeller/manud/node404.html).
  • the polypeptide component of the ATP sensor of the invention comprises sequence corresponding to one of the polypeptides in the above table.
  • Sequence alignment was determined, using structure/sequence features and the salign command of MODELLER (manual page hups./ ! il l i ab o; u inot!ci i c; ⁇ ⁇ ⁇ n dc i ? h s ;n i ).
  • p 70 80 90 100 110 120 130 4futA AELVARAGRVANVLVA-RGLQVGDRVAAQTE- SVEALVLYLATVRAGGVYLPLNTAYTLHELDYFITD 3c5eA RELSENSQQAANVLSGACGLQRGDRVAWLPRVPEWWLVILGCIRAGLIFMPGTIQMKSTDILYRLQM 3vnqA GRLDAWSDAVARTLLA-EGVRPGDRVALRMSPGAEAIVAILAILKCGAAYVPVDLRNPVSRSDFILAD 4g36A AEYFEMSVRLAEAMKR-YGLNTNHRIWCSENSLQFFMPVLGALFIGVAVAPANDIYNERELLNSMNI 2v7bA GELEERARRFASALRT-LGVHPEERILLVMLDTVALPVAFLGALYAGWPWANTLLTPADYVYMLTH 3fccA KQLKEDSDALAHWISS-EYPDDRSPIMVYGHMQPEMIIN
  • pos 140 150 160 170 180 190 200 4futA AEP-IWCDPS RDGIAAIAAVGATVETLGPDGR GSLTDAAAGAS EAFATIDRGA 3c5eA SKAKAIVAGDEVIQEVDTVASECPSLRIKLLVS-EKSCDGWLNFKKLLNEAS TTHHCVETGS 3vnqA SGASALIG--EP HEGCA VTRWRT AAVAECKD--A E APGPG 4g36A SQPTWFVSKKGLQKILNVQKKLPIIQKII IMDSKTDYQGFQSMYTFVTSHLPPGFNEYDFVPESFDR 2v7bA SHARAVIASGALVQNVTQALESAG- - CQLIVSQP LAPLFEELIDAAA PAAKAAATGC 3fccA SGAKLLLSATAV TVTDL PVRIVSE DNLKDIFFTHK GNTPNPEHAVK 2dlqA SKPTIVFSSKKGLDK
  • pos 280 290 300 310 320 330 340 4futA LFARGSMIFLP FDPD- ILDLMA- -RATVLMGVPTFYTRLLQSPRLT-ETTGHMRLFISGSAPLL 3c5eA WALGACTFVHLLP-KFDPLVILKTLSSYPIKSMMGAPIVYRMLLQQ-DLSSYKFPHLQNCVTVGESLL 3vnqA FSTGAELWLPHWAARTPEQYLAVIIDRGVTVINQTPTAFLALTEAAVRGGRDVSGLRYVIFGGEKLT 4g36A LICGFRWLM- -Y-RFEEELFLRSLQDYKIQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLS 2v7bA LSVGATAILMA-E-RPTADAIFARLVEHRPTVFYGVPTLYANMLVSPNLPARADVAIRICTSAGEALP 3fccA LVTGGTLWAIDKDMIARPK
  • the RMSD was calculated for the same set of ANL superfamily members as above with RpMatB, using the align command in PyMOL
  • the polypeptide component of the ATP sensor of the invention comprises sequence corresponding to one of the polypeptides in the above table.
  • Rhodopseudomonas palustris Appl Environ Microbiol 78(18): 6619-6629.
  • the concentration range of ATP measurable with a fluorescent reagentless biosensor, an adduct of two tetramethylrhodamines with MatB from Rhodopseudom onas palustris, has been increased. Mutations were introduced into the binding site to modify ATP binding but maintain the concomitant fluorescence signal. Using this signal, the effect was monitored for mutations in different parts of the binding site. Out of these, three variants were characterized, each with a single extra mutation in the phosphate-binding loop.
  • T167A and T303A Two variants (T167A and T303A) weakened the binding, changing the dissociation constant from the parent's 6 ⁇ to 123 ⁇ and 42 ⁇ , respectively but having a fluorescence change of ⁇ 3-fold on ATP binding.
  • Kinetic measurements showed that the main effect of these mutations was as an increase in dissociation rate constants.
  • These variants widen the range of ATP concentration that can be measured readily by this biosensor to > ⁇ ⁇ .
  • S170A decreases the dissociation constant of ATP to 3.6 ⁇ and has a fluorescence change of 4.2 on binding ATP. This variant also shows while decreased ADP affinity, thereby increasing selectivity of ATP over ADP to >200-fold.
  • Plasm ids Plasmid pRhoRpMatB (plasmid pTEVs containing the coding sequence of RpMatB (Genbank accession number CAE25665.1) with the point mutations C106A, R286C, Q457C, K488A and an N-terminal His 6 -tag) was as described, and now termed pTEV5_ RpMatB_i (Vancraenenbroeck and Webb 2015). The QuikChange site- directed mutagenesis protocol (Stratagene) was used for single-site mutations of the pRhoRpMatB plasmid. Plasmids were sequenced (GATC Biotech) to confirm the presence of the mutations.
  • pTEV5_ RpMatB_2 The three variant plasmids, containing additional S170A, T167A or T303A mutations, are termed pTEV5_ RpMatB_2, pTEV5_ RpMatB _3, and pTEV5_ RpMatB_4 respectively.
  • RpMatB variants labeled with tetram ethylrhodam ine .
  • Protein expression in Escherichia coli, purification and labeling with tetramethylrhodamine were as described (Vancraenenbroeck and Webb 2015).
  • concentrations of labeled RpMatB variants were determined using the extinction coefficient of the protein at 280 nm, calculated from the sequence via Expasy Protparam (Wilkins, Gasteiger et al. 1999) and the extinction coefficients of tetramethylrhodamine: (31000 M 1 cm 1 ) and (52000 M 1 cm 1 ) (Corrie and Craik 1994)
  • Absorbance and fluorescence m easurem ents Absorbance was measured on a Jasco V-550 UV-Vis Spectrophotometer. Fluorescent measurements were obtained on a Cary Eclipse spectrofluorometer (Agilent Technologies), using a 3-mm pathlength quartz cuvette (Hellma), unless otherwise mentioned. Excitation and emission slits were set at 5 nm. Protein and nucleotide concentrations and buffer conditions are given in the figure legends. For titrations with Rho-MatB variants, excitation was at 553 nm, emission at 571 nm.
  • the biosensor suitably has a C106A mutation to eliminate background labeling at that cysteine and suitably has a K488A mutation to block the adenylation half-reaction of ATP and malonate to malonyl-AMP and pyrophosphate.
  • the resulting protein adduct termed Rho-MatB had essentially no enzyme activity, but bound ATP with a 3 ⁇ 4 of 6 ⁇ .
  • This ATP biosensor, termed Rho- MatB couples ATP binding to a 3.7-fold increase in fluorescence intensity and measures ATP concentrations in the low micromolar range. Of importance is the fact that this biosensor is greatly selective for ATP over ADP.
  • Rho-MatB to change the affinity for ATP, while maintaining a significant fluorescence change on ATP binding. In this way the measurable range ATP concentration has been changed. If the affinity is decreased to allow measurements of higher ATP concentrations, a low, sub-stoichiometric concentration of biosensor can be used, minimizing biosensor usage (Solscheid, Kunzelmann et al. 2015). Alternatively, a biosensor for measurements of lower ATP concentrations by increasing the affinity of Rho-MatB for ATP may be increase sensitivity over the parent biosensor.
  • the method chosen to change affinity was to mutate amino acids identified from the crystal structure as interacting with ATP (Crosby, Rank et al. 2012). Usually these looked unlikely to affect the lid closure, that is they are not positioned at the hinge or involved in interactions across the cleft, nor might they affect the rhodamines directly. In doing this, there was essentially a survey of the active-site amino acids and the effect of (mainly) alanine mutations on ATP affinity, readily measured using the rhodamine fluorescence signal. This survey resulted in three new variants, one tighter and two weaker in ATP binding, potentially resulting in the ability to measure ATP from sub- micromolar to >ioo ⁇ .
  • Rho-MatB's function To examine the effects of substituting these residues on Rho-MatB's function, the individual residues were mutated to alanine on a background of (His 6 /Cio6A/R286C/Q457C/K488A)RpMatB (Vancraenenbroeck and Webb 2015). In addition two variants had the T167S and T303S mutations, so potentially still allowing binding to the triphosphate of ATP. The mutants were expressed and purified as for the parent RpMatB. Only variants that gave reasonable expression were continued to purification and labeling with 5-iodoactetyltetramethylrhodamine (5-IATR).
  • 5-iodoactetyltetramethylrhodamine 5-IATR
  • Table C Fluorescence change and affinity for ATP binding to variants of Rho-MatB.
  • Rho-MatB is the protein without any binding site mutations.
  • the Table shows that the mutations that least perturbed the fluorescence change on ATP binding were all located near the triphosphate. However, the mutations that caused the greatest decrease in affinity (T163A, T166A, T167A, S170A) were all in motif A3, the phosphate binding loop. This suggests that changes to this loop decreases binding but still allows the full conformation change, which controls the interaction between rhodamines and therefore the fluorescence change. In contrast, all the mutations that cause almost complete loss of the fluorescence change are close to the adenosine.
  • T167A, S170A and T303A Three variants that retained large changes in fluorescence signals but had potentially useful changes in binding properties (T167A, S170A and T303A) were chosen for further study. They were purified further and their properties were measured and are described in detail.
  • Rho-MatB Rho-MatB and other proteins labeled with two rhodamines (Chambers, Kajiwara et al. 1974; Hamman, Oleinikov et al. 1996; Okoh, Hunter et al. 2006; Kunzelmann and Webb 2010; Vancraenenbroeck and Webb 2015).
  • Table D Fluorescence changes, dissociation constants and rate constants for ATP binding to variants of Rho-MatB.
  • ADP is likely to be present also and may be at relatively high concentration.
  • calibration curves were constructed at different levels of ADP (Figure 24).
  • the total concentration of nucleotide (ATP + ADP) was held constant, to mimic partial interconversion of the two nucleotides.
  • the response was approximately linear up to a concentration of ATP, 50% of the 3 ⁇ 4-value: of course, this is the first part of the binding curve in Figure 23.
  • the fast phase ( Figure 25) had a rate constant that increased linearly with ATP concentration and was interpreted as binding.
  • the association rate constant from the gradient only varied little.
  • the dissociation rate constant could be estimated from the intercept and is the main difference between variants.
  • the T170A variant strengthens binding. This amino acid does not appear to interact with the triphosphate directly, unlike the hydrogen bonding of T167A and T303A. ATP and ADP binding was measured, as described above for the other variants.
  • the mutations in the binding site give us insights into the contributions of specific residues to ATP binding.
  • the fluorescence labelling provides a signal to measures the effect of the amino acid side chain on the affinity of a protein-ligand complex.
  • the effect of three mutations in the phosphate binding loop were studied in detail, as these gave desirable modifications to the biosensors of the invention for measuring ATP, such as the exemplary Rho-MatB.
  • T167A and T303A decrease the affinity and so provide biosensors in the tens of micro molar ATP, up to >ioo ⁇ . These can be used substoichiometrically, for example at ⁇ ⁇ , whereby the concentration of ATP is directly correlated to the fraction of the Rho-MatB in the high fluorescence, ATP-bound state.
  • S170A increased ATP affinity, producing a more sensitive probe for ATP with a larger selectivity for ATP over ADP than the parent biosensor.
  • the greater sensitivity comes from a combination of tighter binding and larger fluorescence enhancement.

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Abstract

The invention relates to an ATP binding molecule comprising a polypeptide which undergoes a conformational change from a first conformation to a second conformation upon binding of ATP, said polypeptide comprising amino acid sequence corresponding to at least amino acids 5 to 497 of SEQ ID NO:1, said polypeptide having at least 21% sequence identity to SEQ ID NO:1, said polypeptide having a value of RMSD <4 Å relative to RpMat B in the ATP-bound conformation, wherein said polypeptide comprises a cysteine residue for attachment of at least one reporter moiety, and comprises at least one reporter moiety attached thereto, wherein said cysteine residue for attachment of at least one reporter moiety is positioned such that said reporter moiety undergoes a change in fluorescence upon changing from said first conformation to said second conformation. The invention also relates to use of such a sensor, and to methods for assay of ATP.

Description

ATP SENSOR
BACKGROUND TO THE INVENTION ATP is an intracellular energy source, for example involved in active transport, cell motility and biosynthesis. It is also an important extracellular signalling agent in neurotransmission (Burnstock 2012) and inflammation (Idzko, Ferrari et al. 2014).
ATP is generated through several pathways such as glycolysis, the Krebs cycle and oxidative phosphorylation. This makes it an important assay target and monitoring.
ATP production is widely used to measure enzyme activity in biochemical and cell- based applications. Various ATP assays are known in the art.
However some of those, such as radioisotope assays, are not continuous, which is a problem. Coupled-enzyme assays, including the luciferase-luciferin system (Patergnani, Baldassari et al. 2014), require several reagents, which is a drawback.
Others, like aptamer-based methods (Feng, Dai et al. 2014), require a long incubation time, which is a disadvantage.
Some, like organic probes (Zhou, Xu et al. 2011), show low selectivity for ATP over other nucleotides and only give a color change and no concentration-dependent readout, which limits their usefulness. Others such as (micro)electrode biosensors (Dale and Frenguelli 2012) measure ATP indirectly, which risks introducing errors.
The present invention seeks to overcome problem(s) associated with the art. SUMMARY OF THE INVENTION
A fluorescent, reagentless biosensor for ATP is described. Such biosensors for a target molecule are a single molecular species that consists minimally of a recognition element and a reporter. In this case, the recognition element is a protein that interacts with the target, ATP, namely an ANL superfamily protein. Most suitably the ANL superfamily protein malonyl-coenzymeA synthetase from Rhodopseudom onas palustris (RpMatB) is used. Amino acid sequence derived from this protein is coupled covalently to reporter fluorophore(s) to give a fluorescence change on ATP binding.
In more detail, a fluorescent reagentless biosensor for ATP was developed based on malonyl-coenzyme A synthetase from Rhodopseudom onas palustris (RpMatB) as the protein scaffold and recognition element. In one embodiment two 5- iodoacetamidotetramethylrhodamines were covalently bound to RpMatB to provide the readout. This adduct couples ATP binding to a 3.7-fold increase in fluorescence intensity with excitation at 553 nm and emission at 575 nm. It has micromolar sensitivity for ATP and is highly selective for ATP relative to ADP. Its ability to monitor ATP production was demonstrated in a steady-state kinetic assay in which ATP is a product.
Thus in one aspect the invention provides an ATP binding molecule comprising a polypeptide which undergoes a conformational change from a first conformation to a second conformation upon binding of ATP, said polypeptide comprising amino acid sequence corresponding to at least amino acids 5 to 497 of SEQ ID NO:i,
said polypeptide having at least 21% sequence identity to SEQ ID NO:i,
said polypeptide having a value of RMSD <4 A relative to RpMatB in the ATP-bound conformation,
wherein said polypeptide comprises a cysteine residue for attachment of at least one reporter moiety, and comprises at least one reporter moiety attached thereto, wherein said cysteine residue for attachment of at least one reporter moiety is positioned such that said reporter moiety undergoes a change in fluorescence upon changing from said first conformation to said second conformation.
Suitably the RMSD is calculated using the PyMOL program.
Suitably the RMSD is calculated using the "align" command in the PyMOL program. Suitably RpMatB in the ATP-bound conformation is taken as PDB number '4FUT'. Thus suitably said polypeptide has a value of RMSD <4 A relative to PDB number '4FUT' (i.e. RpMatB in the ATP-bound conformation). In one embodiment, suitably sequence identity to SEQ ID NO:i is for the amino acid residues corresponding to those shown in column II of table A. Column II of table A corresponds to 'exposed' residues.
Suitably the polypeptide comprises a first cysteine residue at a position corresponding to a position selected from R286 , A282, D283, H285, E287, S289, A290, K385 , L383, G384, 1386 and D287 of SEQ ID NO: 1,
and wherein the polypeptide comprises a second cysteine residue at a position corresponding to a position selected from
Q457, A456, V458, H460, G461, G461, Q457, H460, L462, G464, Q465, K470 , L466, F469 and M471 of SEQ ID NO: 1.
Suitably the polypeptide comprises a first cysteine residue at a position corresponding to a position selected from R286 and K385 of SEQ ID NO: 1,
and wherein the polypeptide comprises a second cysteine residue at a position corresponding to a position selected from Q457, G461 and K470 of SEQ ID NO: 1.
Suitably the polypeptide comprises a first cysteine residue at a position corresponding to position R286 of SEQ ID NO: 1,
and wherein the polypeptide comprises a second cysteine residue at a position corresponding to position G461 of SEQ ID NO: 1.
Suitably the polypeptide comprises a first cysteine residue at a position corresponding to position K385 of SEQ ID NO: 1,
and wherein the polypeptide comprises a second cysteine residue at a position corresponding to position K470 of SEQ ID NO: 1.
Suitably the polypeptide comprises a first cysteine residue at a position corresponding to position R286 of SEQ ID NO: 1,
and wherein the polypeptide comprises a second cysteine residue at a position corresponding to position Q457 of SEQ ID NO: 1. Suitably said molecule comprises the amino acid sequence of SEQ ID NO: 4, SEQ ID NO:5 or SEQ ID NO:6.
Suitably said molecule further comprises at least two tetramethylrhodamine moieties attached thereto. Suitably each of said at least two tetramethylrhodamine moieties is independently selected from the group consisting of 5-tetramethylrhodamine and 6-tetramethylrhodamine.
In one aspect, the invention relates to an ATP binding molecule as described above wherein said cysteine residue for attachment of a reporter moiety is at a position corresponding to a position selected from E439 , I403, P433, G438, G440, N492 , K491, V493, R495, E496 and T497 of SEQ ID NO: 1.
Suitably said cysteine residue for attachment of a reporter moiety is at a position corresponding to E439 or N492 of SEQ ID NO: 1.
Suitably said molecule comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO:3.
Suitably said molecule further comprises at least one diethylaminocoumarin moiety attached thereto.
Suitably said diethylaminocoumarin moiety is independently selected from the group consisting of (N-[2-(i-maleimidyl)ethyl]-7-diethylaminocoumarin-3-carboxamide and N-[2-(iodoacetamido)ethyl]-7-diethylaminocoumarin-3-carboxamide).
In one aspect, the invention relates to an ATP binding molecule as described above wherein said polypeptide comprises an amino acid other than Threonine at the position corresponding to T167 of SEQ ID NO: 1. Suitably said polypeptide comprises alanine or serine at the position corresponding to T167 of SEQ ID NO: 1, preferably alanine.
In one aspect, the invention relates to an ATP binding molecule as described above wherein said polypeptide comprises an amino acid other than Serine at the position corresponding to S170 of SEQ ID NO: 1. Suitably said polypeptide comprises alanine at the position corresponding to S170 of SEQ ID NO: 1.
In one aspect, the invention relates to an ATP binding molecule as described above wherein said polypeptide comprises an amino acid other than Threonine at the position corresponding to T303 of SEQ ID NO: 1. Suitably said polypeptide comprises alanine or serine at the position corresponding to T303 of SEQ ID NO: 1, preferably alanine. In one aspect, the invention relates to an ATP binding molecule as described above wherein said polypeptide comprises an amino acid other than cysteine at the position corresponding to C106 of SEQ ID NO: l. In one aspect, the invention relates to an ATP binding molecule as described above wherein said polypeptide comprises alanine at the position corresponding to C106 of SEQ ID NO: \. (CIO 6 A)
In one aspect, the invention relates to an ATP binding molecule as described above wherein said polypeptide comprises an active site mutation such that the polypeptide is catalytically inactive for ATP hydrolysis.
In one aspect, the invention relates to an ATP binding molecule as described above wherein said active site mutation comprises an amino acid other than lysine at the position corresponding to K488 of SEQ ID NO: 1.
In one aspect, the invention relates to an ATP binding molecule as described above wherein said polypeptide comprises alanine at the position corresponding to K488 of SEQ ID NO: 1. (K488A)
In one aspect, the invention relates to an ATP binding molecule as described above further comprising the sequence of SEQ ID NO: 8.
In one aspect, the invention relates to a nucleic acid having a nucleotide sequence encoding the polypeptide portion of an ATP binding molecule as described above.
In one aspect, the invention relates to a method for monitoring changes in ATP concentration in a sample comprising contacting said sample with an ATP binding molecule as described above and determining changes in conformation of said ATP binding molecule, wherein changes in conformation of said ADP binding molecule indicate changes in the concentration of ATP in said sample.
Suitably the conformation of said ATP binding molecule is monitored by measurement of changes in fluorescence of a fluorophore comprised by said ATP binding molecule. Suitably the sample comprises divalent Magnesium ion (Mg2+).
In one aspect, the invention relates to use of an ATP binding molecule as described above in the determination of ATP concentration in a sample. In one aspect, the invention relates to an ATP binding molecule comprising a polypeptide which undergoes a conformational change from a first conformation to a second conformation upon binding of ATP, said polypeptide comprising amino acid sequence corresponding to at least amino acids 5 to 497 of SEQ ID NO:i,
said polypeptide having at least 34% sequence similarity to SEQ ID NO:i,
wherein said polypeptide comprises a cysteine residue for attachment of at least one reporter moiety, and comprises at least one reporter moiety attached thereto, wherein said cysteine residue for attachment of at least one reporter moiety is positioned such that said reporter moiety undergoes a change in fluorescence upon changing from said first conformation to said second conformation.
In one aspect, the invention relates to an ATP binding molecule comprising a polypeptide which undergoes a conformational change from a first conformation to a second conformation upon binding of ATP, said polypeptide comprising amino acid sequence corresponding to at least amino acids 5 to 497 of SEQ ID NO:i,
said polypeptide having at least 21% sequence identity to SEQ ID NO:i,
wherein said polypeptide comprises a cysteine residue for attachment of at least one reporter moiety, and comprises at least one reporter moiety attached thereto, wherein said cysteine residue for attachment of at least one reporter moiety is positioned such that said reporter moiety undergoes a change in fluorescence upon changing from said first conformation to said second conformation.
DETAILED DESCRIPTION OF THE INVENTION
The invention provides a single solution to the problem of provision of an ATP biosensor. In one embodiment, the biosensor is singly labelled. In another embodiment, the biosensor is doubly labelled. In the singly labelled embodiment, the single dye molecule attached to the biosensor interacts with the protein, and this interaction affects the signal such as fluorescence. Binding of ATP changes the conformation of the biosensor, and thus alters the interaction the dye molecule with the polypeptide. This ATP bound states exhibits a difference in reporter activity such as fluorescence and therefore serves to read out the event of ATP binding. In a double labelled embodiment, the two dye molecules attached to the biosensor interact with one another, for example via dye stacking. When ATP binds to the biosensor, the conformation changes and this conformation change affects the readout such as fluorescence of the two dye molecules. This change in reporter activity such as fluorescence again reads out the presence of ATP. It must be emphasised that whether the biosensor is singly labelled or doubly labelled as outlined, the invention is based upon the same physical conformation change, and the dye(s) is/are attached to the biosensor in the different embodiments are responding to this same conformational change. Therefore, the principle of the invention is the same regardless of whether the single labelled embodiment or the doubly labelled embodiment is considered.
In other words, the single label or dual labelling approaches both rely on a signal such as fluorescence signal due to the closing of the "lid domain" on ATP binding. The single label detects the movement through interactions with the protein surface. The dual (e.g. rhodamine) label detects the relative position of the two labels, which changes on the closure.
For some applications, a doubly labelled biosensor of the invention is especially suitable. Suitably the biosensor comprises a first label and a second label which can exhibit molecular stacking and wherein the molecular stacking is altered on changing from one conformation to the other. Suitably the first and second labels can exhibit molecular stacking either (a) in the first conformation but not in the second conformation, or (b) in the second conformation but not in the first conformation. In one embodiment the first and second labels exhibit molecular stacking in the first conformation. In one embodiment the first and second labels exhibit molecular stacking in the second conformation.
A doubly labelled biosensor suitably uses rhodamine dye molecules. These embodiments of the invention can produce the largest signal. In addition, dyes such as rhodamine can exhibit superior stability during fluorescence measurements, such as greater stability under irradiation. For example, rhodamine is more photostable than coumarins. For example, rhodamine has a longer wavelength excitation, often making it easier to use. Thus suitably the dye used in the biosensors of the invention is rhodamine.
In arriving at their choice of donor molecule from which to create the sensors of the invention, the inventors faced numerous challenges. One such challenge was the availability of 3-dimensional structures from which to study the candidate polypeptides. For example, two crystal structures for RpMatB were used, an apo conformation and an ATP-bound conformation. Although the published structures were crystal structures, in reality this is a flexible and dynamic protein. In particular the protein exhibits a moving cap feature. However, there are no NMR structures (which tend to show such flexibility) available. This presented challenges which were overcome by the present invention. Considerable search effort was made by the inventors in arriving at the choice of RpMatB and ANL superfamily proteins as suitable sensor molecules.
The choices of suitable sites for dye attachment within the sensors of the invention were intellectually challenging. The inventors overcame numerous problems in arriving at the teachings presented herein.
Without wishing to be bound by theory, it is believed that sensors of the invention may exist in two conformations. It is thought that these conformations may reflect monomelic and dimeric forms of the protein. It appears that the monomeric form is the most effective at ATP binding. Nevertheless, it is clear from the practical experiments provided in the application that whether or not this theory is correct, the sensors perform very well regardless of the precise description of their monomeric/dimeric forms. ANL SUPERFAMILY
Suitably other proteins in the ANL superfamily may be used as ATP sensors according to the invention when appropriately labelled. In more detail, the inventors have studied the structural similarity amongst the ANL superfamily members, and we teach that the information from the exemplary sensor based on RpMatB allows positioning of acceptor amino acids such as cysteines (i.e. cysteine substitutions), and hence reporter moieties (e.g. fluorophores), on other ANL superfamily members that bind ATP and show similar ATP-dependent conformation change. Thus suitably the polypeptide of a sensor of the invention may comprise a structural homologue of RpMatB, such as an ANL superfamily protein.
The term 'ANL superfamily' is well known in the art. In case any guidance is needed, the ANL superfamily of adenylating enzymes contains acyl- and aryl-CoA synthetases, firefly luciferase, and the adenylation domains of the modular Non-Ribosomal Peptide Synthetases (NRPSs). Members of this family catalyse two partial reactions, the initial adenylation of a carboxylate to form an acyl-AMP intermediate, followed by a second partial reaction, most commonly, the formation of a thioester. This is described in more detail in the art, such as in Gulick 2009 (ACS Chem Biol. 2009 October 16; 4(10): 811-827. doi:io.i02i/cb900i56h), which is incorporated herein by reference specifically for the information relating to the ANL superfamily.
In case any further information is helpful, reference is made to the Pfam database. The Pfam database is a large collection of protein families, each represented by multiple sequence alignments and hidden Markov models (HMMs). (The Pfam protein families database: R.D. Finn, A. Bateman, J. Clements, P. Coggill, R.Y. Eberhardt, S.R. Eddy, A. Heger, K. Hetherington, L. Holm, J. Mistry, E.L.L. Sonnhammer, J. Tate, M. Punta Nucleic Acids Research (2014) Database Issue 42:D222-D23o). Regarding the Pfam database, suitably the release number of the database referred to is Pfam 27.0 (March 2013, 14831 families).
The Pfam description of the ANL superfamily (htt : / ' fa^^^
mentions:
"This superfamily consists of enzymes including luciferase, long chain fatty acid Co-A ligase, acetyl-CoA synthetase and various other closely-related synthetases as well as a plant auxin-responsive promoter family. The name ANL derives from from three of the subfamilies - Acyl-CoA synthetases, the NRPS adenylation domains, and the Luciferase enzymes [Gulick AM;, ACS Chem Biol. 2009;4:811-827.: Conformational dynamics in the Acyl-CoA synthetases, adenylation domains of non-ribosomal peptide synthetases, and firefly luciferase.]. Members of this superfamily catalyse the initial adenylation of a carboxylate to form an acyl-AMP intermediate, followed by a second partial reaction, most commonly the formation of a thioester [Gulick AM;, ACS Chem Biol. 200954:811- 827.: Conformational dynamics in the Acyl-CoA synthetases, adenylation domains of non-ribosomal peptide synthetases, and firefly luciferase.]."
In structural terms, the "lid" domain, upon which the conformational change on ATP binding is based, is common to ANL superfamily proteins.
In addition, ANL superfamily members have two partial reactions. Thus in a broad aspect the invention relates to ATP sensors which comprise an ANL superfamily protein. Suitably said ANL superfamily protein comprises variants of RpMatB having amino acid substitutions as described, or comprises RpMatB as exemplified.
Guidance regarding the sequence identity/similarity between ANL superfamily members is presented below.
Suitably sequence identity/ similarity is assesses along the whole length of the amino acid sequence present in the polypeptide component of the ATP sensor, unless otherwise specified.
An example of low sequence homology with RpMatB for proteins in the PDB belonging to the ANL superfamily is for 3DHV which has
- A sequence identity of 21% with RpMatB
- A sequence similarity of 38% (calculated using the BLOSUM62 matrix with a gap penalty of 10.0 and a extend penalty of 0.5).
- An RMSD value of 2.9 A compared to 4FUT (both proteins are in the "closed" ATP- bound conformation and the RMSD value was calculated in PyMOL using the "align" command).
Sequence similarity is well known in the art and takes account of conservative substitutions (see table below). However, more importantly, the ANL superfamily proteins have the same structural fold as calculated via the RMSD (root mean square deviation) value. Suitably the polypeptide ATP sensors of the invention have this fold.
RMSD (Root Mean Square Deviation) is the square root of the mean of the square of the distances between the matched atoms:
RMSD = SQRT[{ SUM(dii)2}/N]
where da is the distance between the 1th atom of structure 1 and the 1th atom of structure 2 and N is the number of atoms matched in each structure. An RMSD is calculated between structures (rather than sequences). The RMSD can be used to compare protein three-dimensional structures. The RMSD is 0 for identical structures, and its value increases as the two structures become more different. RMSD values are considered as reliable indicators of variability when applied to very similar proteins. Generally, a value of RMSD <4 A indicates significant structural similarities.
Different programs give different RMSD values, not because they use a different formula but because they use different cut-off parameters to determine which atoms included in the formula. Therefore, the program and command used to calculate the RMSD value must be taken into account.
For example, PyMOL (used in Example 12) calculates it after cycles of refinement in order to reject structural outliers found during the fit - at least when you use the "align" command. Another program, SuperPose does not follow this procedure. Its RMSD is usually higher than the one calculated via PyMOL (it takes more atoms into account). The skilled worker will be aware of these differences.
Suitably the RMSD is calculated using the PyMOL program.
Suitably RMSD values mentioned herein are calculated using the PyMOL program.
The PyMOL program is available for example from htt : // w . py moi . org/, or from Schrodinger, 101 SW Main Street, Suite 1300, Portland, OR 97204, USA.
Suitably the RMSD is calculated using the "align" command in the PyMOL program. Suitably RMSD values mentioned herein are calculated using the "align" command in the PyMOL program.
RpMatB exists in multiple conformations (see below), so it is important to calculate the RMSD values for the structure pairs that have the same conformation.
Suitably RMSD values mentioned herein are for the ATP bound conformation(s).
Suitably the reference structure is RpMatB in the ATP-bound conformation.
See for example Figure 16 (Supplemental Table 3).
Suitably the polypeptide component of the ATP sensor of the invention is comprises an ANL superfamily protein. Suitably said polypeptide comprises a value of RMSD <4 A relative to RpMatB; more suitably said polypeptide comprises a value of RMSD <3 A relative to RpMatB.
It is straightforward for the skilled worker to identify residues in such protein(s) corresponding to the labelling/substitution positions in RpMatB. In case any further information is required, guidance is provided below, particularly in the examples section.
RpMatB
RpMatB was chosen as the exemplary recognition element of the ATP sensors described because of several properties, including high yield expression and purification, good stability and high affinity and selectivity for ATP. RpMatB belongs to the AMP-forming acyl-coenzymeA synthetase family (PF00501 (Finn, Bateman et al. 2014)) and the ANL superfamily containing acyl- and aryl-coenzymeA synthetases, the adenylation domains of nonribosomal peptide synthetases and firefly luciferase (Gulick 2009). RpMatB catalyzes the conversion of malonate and coenzymeA to malonyl- coenzymeA via a ping-pong mechanism consuming ATP through a malonyl-AMP intermediate. Its products are AMP, pyrophosphate and malonyl-coenzymeA.
RpMatB has been crystallized in two conformations, that is an open form of the apoprotein and a closed form with MgATP bound (Crosby, Rank et al. 2012) (Figure 1). The ligand-binding pocket is between the N- and C-terminal lobes. Upon binding MgATP, the C-terminal lobe rotates ~20° to close the binding cleft. This conformational change was used to create a series of rationally designed RpMatB mutants with cysteine point mutations of surface amino acid residues in order to incorporate thiol-reactive fluorophores.
We describe different design strategies to create a fluorescence signal that can be used to report the ATP concentration. One is based on the introduction of one environmentally sensitive fluorophore, namely diethylaminocoumarin. The other relies on reversible stacked dimer formation between a pair of identical fluorophores, more particular two tetramethylrhodamines. Both fluorophores have been used to develop biosensors. Examples include an inorganic phosphate biosensor based on the E. coli phosphate binding protein (Brune, Hunter et al. 1994, Okoh, Hunter et al. 2006), a single stranded DNA biosensor based on the E. coli single stranded DNA binding protein (Dillingham, Tibbies et al. 2008) and an ADP biosensor based on the bacterial actin homologue, ParM (Kunzelmann and Webb 2009, Kunzelmann and Webb 2010). The exemplary ATP biosensor described herein is an adduct of RpMatB and tetramethylrhodamine, which specifically responds to ATP with a maximum 3.7-fold fluorescence increase. Its sensitivity lies in the micromolar range. Its ability to monitor ATP production was demonstrated with a steady-state kinetic assay to measure the time course of enzymatic ATP production.
In order to introduce the taught mutations into the particular ANL superfamily protein which it is designed to use, then the corresponding sites for mutation should be identified by reference to the exemplary RpMatB sequence (SEQ ID NO: 1).
Suitably the polypeptide of a sensor of the invention comprises amino acid sequence corresponding to RpMatB amino acid sequence, comprising substitutions as described. w ild-type RpMat se quence (accession number: Genbank CAE25665.1)
MNANLFARLFDKLDDPHKLAIETAAGDKI SYAELVARAGRVANVLVARGLQVGDRVAAQTEKSVEALVLY LATVRAGGVYLPLNTAYTLHELDYF I TDAEPKI WCDPSKRDGIAAIAAKVGATVETLGPDGRGSLTDAA AGASEAFAT I DRGADDLAAI LYTSGTTGRSKGAMLSHDNLASNSLTLVDYWRFTPDDVL IHALP IYHTHG LFVASNVTLFARGSMIFLPKFDPDKI LDLMARATVLMGVPTFYTRLLQSPRLTKETTGHMRLF I SGSAPL LADTHREWSAKTGHAVLERYGMTETNMNTSNPYDGDRVP GAVGPALPGVSARVTDPETGKELPRGD I GMI EVKGPNVFKGYWRMPEKTKSEFRDDGFF I TGDLGKI DERGYVHI LGRGKDLVI TGGFNVYPKE IESE I DA MPGWESAVI GVPHADFGEGVTAVWRDKGAT I DEAQVLHGLDGQLAKFKMPKKVIFVDDLPRNTMGKVQ KNVLRETYKD IYK (SEQ ID NO: 1 )
RpMatB polype ptides
The polypeptide components of the molecules of the invention are based on ANL superfamily polypeptide sequences such as the exemplary RpMatB sequence. In particular, amino acid addresses given in the application correspond to the numbering of the RpMatB reference sequence of SEQ ID NO:i. Where truncated or extended forms of RpMatB are used as polypeptides in molecules of the invention (e.g. where a 6his tag is added or where a section of the polypeptide is deleted) then the amino acid numbering should be treated as corresponding to the equivalent section of the full length RpMatB reference sequence and not as an 'absolute' or rigidly inflexible numeric address. By way of explanation, if the description mentions a substitution of K488, this means amino acid 488 of the RpMatB reference sequence of SEQ ID NO: 1. If the polypeptide used is truncated by deletion of the first 10 amino acids, the address given will still be K488 (rather than e.g. K478) - this will be easily understood by the skilled reader to refer to the amino acid of the corresponding context with reference to the full length RpMatB sequence of SEQ ID NO:i, as is conventional in the art. Clearly there are elements of the RpMatB wild type sequence which we teach are important to mutate, such as by substitution, to achieve certain advantages. However, there are also numerous residues which may or may not be mutated depending on operator choice. Clearly there are also numerous residues which should not be mutated in case such mutation would interfere with the function of the polypeptide. Typically it would be expected that if the skilled operator had a concern whether or not a particular residue could be mutated or not, they could make the mutation and then test the resulting polypeptide to ensure that the desired property was retained in the mutated version. However, in order to provide further guidance on this point, the following comments are made:
The exemplary biosensor polypeptide RpMatB has been studied in detail and each residue has been classified as set out in Table A below. The classification is as follows, making use of information on surface exposure:
Buried: Residues in the core of the structure where mutations are likely to affect the function of the sensor.
Exposed: Residues on the surface of the protein. Mutations of these residues are likely to retain the sensor function. Possibly any mutation may be allowed
Intermediate: Change in sensor function when mutating these residues is possible, but may be difficult to predict and conservative mutations are likely to be more successful e.g. routine testing of resulting mutants is particularly preferred when mutating these residues. Most of these are partially buried.
Residues marked with an asterisk (*) designate active site residues. Active site residues may suitably be specifically mutated in order to optimise the sensor functions (such as to impair or eliminate ATP hydrolysis such as for example mutating K488). These are discussed in more detail in the text.
In more detail, the relative accessible surface area (ASA) of a residue is its ASA calculated using DSSP (Kabsch, W. and C. Sander (1983). "Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features." Biopolymers 22(12): 2577-637.) divided by its nominal maximum area as defined by (Chothia, C. (1976). "The nature of the accessible and buried surfaces in proteins." J Mol Biol 105(1): 1-12). Here, a residue is defined as exposed if its relative
ASA is at least 40% of its nominal maximum area. A residue is defined as buried if its relative ASA is less than 10% of its nominal maximum area. Residues marked with asterisk (*) designate active site residues (within 6 A of ATP and Mg2+).
Table A - RpMatB residues
Buried (I) Exposed (II) Intermediate (III)
Numbering Amino acid Numbering Amino acid Amino
Numbering acid
4 N 1 M
2 N
5 L 8 R
6 F 11 D 3 A
7 A 12 K 13 L
16 P
9 L 14 D
10 F 15 D 19 L
20 A 23 T
17 H
21 I 18 K 30 S
22 E 24 A 32 A
29 I 25 A 35 V
31 Y 26 G 36 A
34 L 27 D 37 R
40
38 A 28 K
39 G 33 E 43 N
41 V 46 V 49 G
42 A 47 A 53 G
48 R 54 D
44 V
55 R
45 L 51 Q
60 T
50 L 52 V
62 K
56 V 61 E
57 A 86 A 63 S
58 A 88 T 65 E
59 Q 89 L 84 N
64 V 90 H 85 T
66 A 97 T 87 Y
67 L 100 E 91 E
68 V 102 K 93 D
69 L 108 P 94 Y
70 Y 109 S 98 D
107 D
71 L 111 R
110 K
72 A 112 D
73 T 116 A 113 G
74 V 119 A 115 A
121 V
75 R 120 K
123 A
76 A 122 G
124 T
77 G 130 P
78 G 131 D 125 V
79 V 133 R 134 G
80 Y 138 D 135 S 81 L 141 A 137
82 P 142 G 139 A
83 L 144 S 140 A 92 L 145 E 143 A
95 F 146 A 147 F
96 I 148 A 149 T 99 A 151 D 150 I lOl P 153 G 152 R
103 I 155 D 154 A
104 V 164 S (*) 156 D
105 V 165 G (*) 157 L
106 C 167 T (*) 162 Y 114 I 168 G (*) 163 T (*)
117 I 169 R (*) 170 S (*)
118 A 189 D 171 K (*)
126 E 192 R 174 M
127 T 194 T 178 D
128 L 195 P 182 S
129 G 196 D 185 L 132 G 207 H (*) 186 T 136 L 229 P 188 V
158 A 230 K 190 Y
159 A 232 D 198 V
160 I 234 D 204 P
161 L 235 K 206 Y 166 T (*) 238 D 220 F
172 G 241 A 222 R
173 A 242 R 224 S
175 L 251 T 231 F
176 S 255 R 233 P
177 H 258 Q 244 T
179 N 260 P 249 V
180 L 264 K 250 P
181 A 265 E 254 T
183 N 268 G 257 L
184 S 269 H 259 s 187 L 277 s o 261 R 191 W 278 A (*) 263 T 193 F 279 P C) 266 T 197 D 283 D 271 R
199 L 286 R 281 L
200 I 287 E 282 A
201 H 289 S 294 H
202 A 290 A 298 E (*)
203 L 291 K 301 G (*) 205 I 293 G 302 M (*) 2θ8 T (*) 295 A 303 T (*)
209 H 314 D 304 E (*)
210 G 315 G 312 P
211 L 316 D 313 Y
212 F
317 R 318 V
213 V 320 G 319 P
214 A
327 P 322 V (*)
215 S 328 G 324 P
216 N 330 S 325 A
217 V 336 P 329 V
218 T 337 E 331 A
219 L 338 T 332 R 221 A 340 K 335 D 223 G 341 E 339 G
225 M
343 P 342 L
226 I
344 R 347 I
227 F
345 G 349 M
228 L 346 D 354 G
236 I 353 K 355 P
237 L 359 K 364 M
239 L 363 R 368 T
240 M 365 P 371 E 243 A 366 E 372 F
245 367 K 373 R
246 L 369 K 376 G
247 M 370 S 377 F
248 G 374 D 379 I (*)
252 F 375 D 381 G
253 Y 385 K 382 D (*) 256 L 388 E 386 I 262 L 389 R 387 D 267 T
397 R (*) 390 G 270 M 399 K 393 H
272 L 403 I 394 I (*)
273 F 405 G 395 L
274 I 406 G 396 G (*)
275 S (*) 407 F 398 G
276 G (*) 408 N 400 D 280 L (*) 412 K 401 L
284 T 413 E 410 Y
285 H 416 S 415 E 288 W 417 E 419 D 292 T 420 A 421 M
296 V 422 P 423 G
297 L 425 V 426 E 299 R (»)
433 P 434 H 300 Y (*) 435 A 437 F
305 τ 436 D 439 E
306 Ν 438 G 446 V
307 Μ 447 R 451 A
308 Ν 448 D 459 L
309 Τ 449 K 466 L
310 S 450 G 467 A
311 Ν 452 T 469 F
321 A 454 D 470 K
323 G 455 E 471 M
326 L 456 A 476 I
333 V 457 Q 477 F
334 Τ 460 H 478 V
348 G 461 G 482 P
350 I 463 D 484 N (*)
351 Ε 464 G 489 V
352 V 465 Q 490 Q (*)
356 Ν 468 K 491 K
357 V 473 K 495 R
358 F 474 K 498 Y
360 G 479 D 501 I
361 Υ 480 D 502 Y
362 W 483 R
378 F 485 T
380 Τ 486 M
383 L 487 G
384 G 488 K (*)
391 Υ 492 N
392 V 493 V
402 V 496 E
404 Τ 497 T
409 V 499 K
411 Ρ 500 D
414 I 503 K
418 I
424 V
427 S
428 A
429 V
430 I
431 G
432 V
440 G
441 V
442 Τ
443 A 444
445
453 I
458 V
462 L
472 P
475
481 L
494 L
Buried Residues
A few conservative changes may be possible in the buried region. Suitably residues in the polypeptide part of an ATP sensor molecule of the invention have at least 90% sequence identity to RpMatB (SEQ ID NO:i). Suitably any differences are conservative substitutions. Suitably residues in the polypeptide part of an ATP sensor molecule of the invention have 100% similarity to RpMatB (SEQ ID NO:i). More suitably residues shown as 'buried' in table A are not mutated. Thus suitably residues in the polypeptide part of an ATP sensor molecule of the invention which correspond to RpMatB residues shown as 'buried' in table A are not mutated relative to RpMatB (SEQ ID NO:i). In other words, suitably residues in the polypeptide part of an ATP sensor molecule of the invention shown as 'buried' in table A comprise the same residue as at the corresponding position in RpMatB (SEQ ID NO:i).
Thus in some embodiments, the polypeptide component of the ATP sensor molecule of the invention suitably comprises amino acid sequence having 100% sequence identity to those residues shown as 'buried' in table A. Interm ediate Residues
Suitably residues shown as 'intermediate' in table A may be mutated. Thus suitably residues in the polypeptide part of an ATP sensor molecule of the invention which correspond to RpMatB residues shown as 'intermediate' in table A may be mutated relative to RpMatB (SEQ ID NO:i). In other words, suitably residues in the polypeptide part of an ATP sensor molecule of the invention shown as 'intermediate' in table A may comprise a different residue (or no residue) from the corresponding position in RpMatB (SEQ ID NO:i).
Suitably a biosensor of the invention has at least 60% sequence identity to SEQ ID NO: 1. In some embodiments, the polypeptide component of the ATP sensor of the invention suitably comprises amino acid sequence having at least 60% sequence identity to those residues shown as 'intermediate' in table A. In some embodiments, the polypeptide component of the ATP sensor of the invention suitably comprises amino acid sequence having at least 68% sequence identity to those residues shown as 'intermediate' in table A, suitably least 70% sequence identity, suitably least 74% sequence identity, suitably least 78% sequence identity, suitably least 82% sequence identity, suitably least 86% sequence identity, suitably least 90% sequence identity, suitably least 94% sequence identity, suitably least 98% sequence identity to those residues shown as 'intermediate' in table A.
However, it should be noted that these 'intermediate' residues are in fact partially buried. Thus, suitably these 'intermediate' residues are only mutated by substitution with a conservative residue relative to RpMatB. In other words, suitably the non- identical residues noted above comprise only conservative substitutions relative to the corresponding residue in RpMatB.
Unless otherwise apparent from the text, mentions of 'sequence homology' suitably refer to sequence identity. However, occasionally it is more appropriate to refer to sequence similarity. Sequence similarity takes account of sequence identity and also takes account of conservative substitutions (i.e. non-identical residues but where the residue is similar or conserved compared to the original residue). Assessing sequence similarity is well known in the art. Examples are provided in the examples section. In case any further guidance is needed, suitably the following parameters are used in the algorithm for calculating sequence similarity: BLOSUM62 matrix, gap penalty 10.0, gapextend penalty 0.5; more suitably BLOSUM62 matrix, gapopen 10.0, gapextend 0.5, endopen 10.0, endextend 0.5, pairwise alignment. Suitably the polypeptide component of the ATP sensor of the invention has at least 34% sequence similarity to SEQ ID NO: 1, suitably at least 38% sequence similarity, suitably at least 40% sequence similarity, suitably at least 50% sequence similarity, suitably at least 60% sequence similarity, suitably at least 65% sequence similarity, suitably at least 70% sequence similarity, suitably at least 75% sequence similarity, suitably at least 80% sequence similarity, suitably at least 85% sequence similarity, suitably at least 90% sequence similarity, suitably at least 95% sequence similarity, suitably at least 98% sequence similarity, suitably at least 99% sequence similarity, most suitably 100% similarity to SEQ ID NO: 1.
Conservative substitutions may be made for example according to the table below. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other:
Figure imgf000022_0001
More suitably residues shown as 'intermediate' in table A are not mutated. Thus suitably residues in the polypeptide part of an ATP sensor of the invention which correspond to residues shown as 'intermediate' in table A are not mutated relative to RpMatB (SEQ ID NO:i). In other words, suitably residues in the polypeptide part of an ATP sensor of the invention shown as 'intermediate' in table A comprise the same residue as at the corresponding position in RpMatB (SEQ ID NO:i).
Exposed Residues
Suitably residues shown as 'exposed' in table A (i.e. column II of table A) may be mutated. Thus suitably residues in the polypeptide part of an ATP sensor of the invention which correspond to residues shown as 'exposed' in table A may be mutated relative to RpMatB (SEQ ID NO:i). In other words, suitably residues in the polypeptide part of an ATP sensor of the invention shown as 'exposed' in table A may comprise a different residue (or no residue) from the corresponding position in RpMatB (SEQ ID NO:i).
In some embodiments, the polypeptide component of the ATP sensor of the invention suitably comprises amino acid sequence having at least 21% sequence identity to those residues shown as 'exposed' in table A, suitably at least 30% sequence identity, suitably at least 40% sequence identity, suitably at least 50% sequence identity, suitably at least 60% sequence identity, suitably at least 65% sequence identity, suitably at least 70% sequence identity, suitably at least 75% sequence identity, suitably at least 80% sequence identity, suitably at least 85% sequence identity, suitably at least 90% sequence identity, suitably at least 95% sequence identity, suitably at least 98% sequence identity, suitably at least 99% sequence identity, most suitably 100% identity to those residues shown as 'exposed' in table A.
AMINO ACID RESIDUES
For dye conjugation we teach specific examples of specific amino acid residues for dye attachment for the sensor molecules of the invention. Details of these are provided herein. It may also be possible to use other residues in the vicinity of those specified for dye attachment. By "in the vicinity" we mean a near neighbour of the specified amino acid. A near neighbour of the specified amino acid may mean an adjacent amino acid i.e. the amino acid before or the amino acid after the one specified. Alternatively, neighbouring amino acid may be used. A neighbouring amino acid may refer to an amino acid two residues either side of the residue specified, for example if the residue specified is 398 then residues 400 and 396 would be considered neighbouring amino acids. For example, if amino acid 398 was specified, then residues 399 and 397 would be considered near neighbours. Alternatively, a further neighbour of the amino acid may be specified, for example, if amino acid 398 is taught for attachment then a further neighbour might be three amino acid residues away such as residue 401 or residue 395. More distant amino acids may be used if desired. In all cases, it is advisable to check the performance of the sensor using the assays as taught herein.
More suitably, an amino acid which is "in the vicinity" of a specified amino acid is one which is present in a physically adjacent 3-dimensional space. For example, an amino acid specified on a part of an a helix will have a neighbouring amino acid in the vicinity such as the residue at the same position on the next turn of that a helix. For example, amino acid 457 is on one side of an a helical section of the protein; thus amino acid 461 is a neighbouring amino acid in the vicinity of amino acid 457 since it is on the corresponding side of the next turn of the same a helix. Thus, when considering the 3- dimensional structure of the protein, an amino acid which might seem "distant" in terms of the number of intervening residues may actually be a neighbouring amino acid in the vicinity of the specified amino acid if it is close in 3-dimensional space. Amino acid residues close in space to those exemplified for labelling (see below and in Examples section) may also be suitable for labelling. Examples of surface accessible residues & neighbouring amino acids are now discussed to aid understanding.
Based on the exemplified cysteine substitution (mutation) sites, Table 2 below shows examples of amino acids which are neighbouring in sequence or in space. In the latter case, this represents amino acids in a similar location on the next turn of a helix, on the same side of a beta-sheet or on an adjacent sheet of a beta-plate.
Table 2: The neighbouring residues in sequence and space for the amino acid mutations of the diethylaminocoumarin (MDCC and IDCC) and tetramethylrhodamine (5-ATR and 6-ATR) labeled RpMatB cysteine mutant sensor molecules:
Figure imgf000024_0001
These are further shown within the sequence of MatB and in structural representations below. Suitably the or each reporter moiety (such as fluorophore) is attached to the polypeptide via an amino acid residue corresponding to one or more of those listed in the above table.
Exem plary Sensors of the Invention The following variants provide a good signal (see Examples & Table 1), with a minimum of 60% increase in fluorescence between apo and ATP-bound form (relative to which of these forms had lower fluorescence):
5 Single Reporter (fluorophore):
MDCC-His6-RpMatB C106A/K488A/N492C
IDCC-His6-RpMatB C106A/K488A/N492C
MDCC-His6-RpMatB C106A/E439C/K488A 0 Dual Reporter (double fluorophore):
(6-ATR)2-His6-RpMatB C106A/R286C/G461C/K488A
(6-ATR)2-His6-RpMatB C106A/K385C/K470C/K488A
(6-ATR)2-His6-RpMatB C106A/R286C/Q457C/K488A
(5-ATR)2-His6-RpMatB C106A/R286C/Q457C/K488A
5
Exemplary Sensor Sequences based on RpMat
(Substitutions (Mutations) shown in bold)
0
Single Label (Single cysteine):
RpMatB C106A/K488A/N492C SEQ ID NO: 2
5 MNANLFARLFDKLDDPHKLAIETAAGDKI SYAELVARAGRVANVLVARGLQVGDRVAAQTEKSVEALVLY LATVRAGGVYLPLNTAYTLHELDYFITDAEPKI WADPSKRDGIAAIAAKVGATVETLGPDGRGSLTDAA AGASEAFATIDRGADDLAAILYTSGTTGRSKGAMLSHDNLASNSLTLVDYWRFTPDDVLIHALPIYHTHG LFVASNVTLFARGSMIFLPKFDPDKILDLMARATVLMGVPTFYTRLLQSPRLTKETTGHMRLFI SGSAPL LADTHREWSAKTGHAVLERYGMTETNMNTSNPYDGDRVP GAVGPALPGVSARVTDPETGKELPRGDIGMIO EVKGPNVFKGYWRMPEKTKSEFRDDGFFITGDLGKIDERGYVHILGRGKDLVITGGFNVYPKEIESEIDA MPGWESAVIGVPHADFGEGVTAVWRDKGATIDEAQVLHGLDGQLAKFKMPKKVIFVDDLPRNTMGAVQ KCVLRETYKDIYK
RpMatB C106A/E439C/K488A SEQ ID NO: 3
5
MNANLFARLFDKLDDPHKLAIETAAGDKI SYAELVARAGRVANVLVARGLQVGDRVAAQTEKSVEALVLY LATVRAGGVYLPLNTAYTLHELDYFITDAEPKI WADPSKRDGIAAIAAKVGATVETLGPDGRGSLTDAA AGASEAFATIDRGADDLAAILYTSGTTGRSKGAMLSHDNLASNSLTLVDYWRFTPDDVLIHALPIYHTHG LFVASNVTLFARGSMIFLPKFDPDKILDLMARATVLMGVPTFYTRLLQSPRLTKETTGHMRLF I SGSAPLO LADTHREWSAKTGHAVLERYGMTETNMNTSNPYDGDRVP GAVGPALPGVSARVTDPETGKELPRGDIGMI EVKGPNVFKGYWRMPEKTKSEFRDDGFFITGDLGKIDERGYVHILGRGKDLVITGGFNVYPKEIESEIDA MPGWESAVIGVPHADFGCGVTAVWRDKGATIDEAQVLHGLDGQLAKFKMPKKVIFVDDLPRNTMGAVQ KNVLRETYKDIYK
Dual Label (Two cysteines): RpMatB C106A/R286C/G461C/K488A SEQ ID NO: 4
MNANLFARLFDKLDDPHKLAIETAAGDKISYAELVARAGRVANVLVARGLQVGDRVAAQTEKSVEALVLY LATVRAGGVYLPLNTAYTLHELDYFITDAEPKIWADPSKRDGIAAIAAKVGATVETLGPDGRGSLTDAA AGASEAFATIDRGADDLAAILYTSGTTGRSKGAMLSHDNLASNSLTLVDYWRFTPDDVLIHALPIYHTHG LFVASNVTLFARGSMIFLPKFDPDKILDLMARATVLMGVPTFYTRLLQSPRLTKETTGHMRLFISGSAPL LADTHCEWSAKTGHAVLERYGMTETNMNTSNPYDGDRVPGAVGPALPGVSARVTDPETGKELPRGDIGMI EVKGPNVFKGYWRMPEKTKSEFRDDGFFITGDLGKIDERGYVHILGRGKDLVITGGFNVYPKEIESEIDA MPGWESAVIGVPHADFGEGVTAVWRDKGATIDEAQVLHCLDGQLAKFKMPKKVIFVDDLPRNTMGAVQ KNVLRETYKDIYK
RpMatB C106A/K385C/K470C/K488A SEQ ID NO: 5
MNANLFARLFDKLDDPHKLAIETAAGDKISYAELVARAGRVANVLVARGLQVGDRVAAQTEKSVEALVLY LATVRAGGVYLPLNTAYTLHELDYFITDAEPKIWADPSKRDGIAAIAAKVGATVETLGPDGRGSLTDAA AGASEAFATIDRGADDLAAILYTSGTTGRSKGAMLSHDNLASNSLTLVDYWRFTPDDVLIHALPIYHTHG LFVASNVTLFARGSMIFLPKFDPDKILDLMARATVLMGVPTFYTRLLQSPRLTKETTGHMRLFISGSAPL LADTHREWSAKTGHAVLERYGMTETNMNTSNPYDGDRVPGAVGPALPGVSARVTDPETGKELPRGDIGMI EVKGPNVFKGYWRMPEKTKSEFRDDGFFITGDLGCIDERGYVHILGRGKDLVITGGFNVYPKEIESEIDA MPGWESAVIGVPHADFGEGVTAVWRDKGATIDEAQVLHGLDGQLAKFCMPKKVIFVDDLPRNTMGAVQ KNVLRETYKDIYK
RpMatB C106A/R286C/Q457C/K488A SEQ ID NO: 6
MNANLFARLFDKLDDPHKLAIETAAGDKISYAELVARAGRVANVLVARGLQVGDRVAAQTEKSVEALVLY LATVRAGGVYLPLNTAYTLHELDYFITDAEPKIWADPSKRDGIAAIAAKVGATVETLGPDGRGSLTDAA AGASEAFATIDRGADDLAAILYTSGTTGRSKGAMLSHDNLASNSLTLVDYWRFTPDDVLIHALPIYHTHG LFVASNVTLFARGSMIFLPKFDPDKILDLMARATVLMGVPTFYTRLLQSPRLTKETTGHMRLFISGSAPL LADTHCEWSAKTGHAVLERYGMTETNMNTSNPYDGDRVPGAVGPALPGVSARVTDPETGKELPRGDIGMI EVKGPNVFKGYWRMPEKTKSEFRDDGFFITGDLGKIDERGYVHILGRGKDLVITGGFNVYPKEIESEIDA MPGWESAVIGVPHADFGEGVTAVWRDKGATIDEACVLHGLDGQLAKFKMPKKVIFVDDLPRNTMGAVQ KNVLRETYKDIYK
As will be clear from the text, these exemplary sequences may have additions/deletions (e.g. N- or C- terminal truncations) or other substitutions as described.
SEQ ID NO: 7 (see Figure 17) shows exemplary sequence annotated to illustrate exemplary substitutions which may be used in the invention. Suitably these exemplary sequences may further comprise an N-terminal addition MSYYHHHHHH DYDIPTSENL YFQGAS (SEP ID NO: 8 ) added directly before the first Methionine of the sequences above. This N-terminal addition comprises a 6His tag useful in purification. TRUNCATIONS /INSERTIONS /DELETIONS
In a broad aspect the invention relates to an ANL superfamily polypeptide for use as an ATP sensor molecule or ATP binding molecule. Such an ANL superfamily polypeptide may be full length (i.e. comprising all 503 amino acid residues corresponding to SEQ ID NO:i (whether or not substitutions relative to SEQ ID NO:i are made in the particular amino acids present)) or truncated. Suitably truncated forms are those which lack a small number of amino acid residues from the N- or C- terminus of the polypeptide relative to wild type. Suitably a small number is 10 or fewer.
In more detail, ANL superfamily proteins such as RpMatB have flexible C- and N- terminal ends. We teach that small truncations might be made at either or both ends. Suitably some or all of the amino acids from these flexible sections may be deleted without adversely affecting the remaining structure and hence retaining sensor function.
In more detail, N-terminal amino acids which may be deleted include those corresponding to Ml, N2, A3, N4 of SEQ ID NO: 1. In more detail, C-terminal amino acids which may be deleted include those corresponding to E498, K499, D500, 1501, Y502, K503 of SEQ ID NO: 1.
In case it is necessary to check the sensor function after such deletion(s), the skilled worker may simply test the sensor as outlined herein.
Similarly, small insertions or deletions may be made in the protein without disrupting function, in particular insertions or deletions are suitably not made in the region of the reporter/dye attachment points, nor in the ATP binding section of the protein. Suitably insertions or deletions are suitably not made in secondary structure elements of the polypeptide such as alpha helices or beta sheets. Suitably insertions/deletions are not made in dual labelled polypeptides in the section of the polypeptide between the dye attachment points, so as to preserve the dye spacing.
TAGS
It is often useful to tag proteins of the invention, for example to facilitate their purification after recombinant production.
Suitably tags may be placed at the extreme C-terminus or the extreme N-terminus of the sensor molecule.
Most suitably tags are placed at the N-terminus of the protein. Suitably one tag per protein molecule is used. Multiple tags per protein molecule may be used if desired, including multiple copies of the same tag or two or more different tags, for example it may be desirable to use a 6 His-tag for purification and in addition to use a Myc-tag for detection.
Tags may be removed from the sensor protein, for example by proteolytic cleavage, or may be retained on the sensor protein during use. A hexahistidine tag (6his) may be added to the polypeptide part of the ATP binding molecule of the invention to simplify purification; most suitably a N-terminal hexahistidine tag is used. 6 His is a particularly useful tag for purification on nickel substrates. However, any suitable tag known in the art may be used. Alternatively, the sensor molecule of the invention may be tagless. Tagless purification (if needed) is well known in the art.
BACKBONE MUTATIONS ANL superfamily proteins may have catalytic activity. For example, RpMatB may consume ATP when carrying out its enzymatic activity. However, RpMatB has no significant ATPase activity (forming ADP and Pi). The reaction for RpMatB is:
-ATP + malonate to malonyl-AMP + PPi
-Malonyl-AMP + CoASH to Malonyl-CoA and AMP.
It is possible to use sensors of the invention with an intact catalytic activity. In this case, it is important that no substrates of the enzyme are present in the assay mixture. The reason is that if substrates are present, this will drive ATP consumption and may distort the data provided by the sensor. For this reason, suitably the sensor molecules of the invention are catalytically inactive. Suitably the sensor molecules of the invention do not comprise ATPase activity. Suitably the sensor molecules of the invention are catalytically inactive for ATP consumption.
The sensor molecule may be catalytically inactivated by mutation of the active site. For example, suitably the sensor of the invention is mutated to render it catalytically inactive. When the sensor comprises an ANL superfamily member such as RpMatB, suitably a K488 mutation is present. In other words, suitably the amino acid corresponding to the wild type K488 is changed to any amino acid other than K. Suitably K488V may be used. Suitably K488A may be used. Thus, suitably the sensor of the invention comprises K488X1, wherein X1 is not K. Suitably the sensor of the invention comprises K488V. Most suitably the sensor of the invention comprises K488A.
The conjugation of dye to the polypeptide of interest may target naturally occurring cysteine residues. To this end, it may be useful to remove unwanted cysteine residues by a process of mutation in order to eliminate background signal. When the sensor of the invention comprises an ANL superfamily member such as RpMatB, suitably cysteine 106 of the wild type protein is mutated to be other than cysteine. For example, suitably the sensor of the invention comprises a C106X2 mutation, wherein X2 is not C. Suitably the sensor of the invention comprises a C106A mutation. Background signal from C106 may be as high as 6%. Therefore, C106X2 (where X2 is not C) mutants such as C106A provide the advantage of eliminating this background signal.
A sensor which is wild type at position 106 i.e. comprising C106 may still be used - in this case the values collected should be adjusted for any background signal as necessary.
MANUFACTURE AND PREPARATION
The polypeptide components of the ATP sensor molecules of the invention may be produced by standard recombinant techniques, such as creating a nucleic acid encoding the amino acid sequence of the polypeptide, and then expressing the polypeptide in a host such as E.coli. Alternatively an in vitro translation may be used. Alternatively the polypeptide itself may be chemically synthesised.
The polypeptide(s) may be purified by any suitable method known in the art, such as 6His tagging the protein then purification using Ni-NTA beads.
To introduce the desired amino acid substitutions into the polypeptide of the invention, any suitable technique may be used such as site directed mutagenesis. For example, mutant PCR primers or oligonucleotides containing the desired nucleotide sequence may be annealed to a template and ligated, extended or amplified to produce a mutated nucleotide sequence encoding the desired substitution. Alternatively the desired nucleotide sequence may be synthesised chemically. Exemplary techniques are presented in the Examples below. REPORTER MOIETIES / DYES
Suitably the ATP binding molecules of the invention comprise at least one reporter moiety attached thereto. The reporter moiety may be any suitable chemical group or structure capable of reading out change(s) in the conformation of said ATP binding molecule. Most suitably the reporter moiety comprises one or more fluorophore(s) such as coumarin or rhodamine.
Reporter moieties used in the invention can give various signals, but preferred labels are luminescent labels. Luminescent labels include both fluorescent labels and phosphorescent labels. However, the use of other labels is envisaged. For example, electrochemical labels could be used wherein alteration in the environment of the labels will give rise to a change in redox state. Such a change may be detected using an electrode. Most suitably fluorescent labels which may be excited to fluoresce upon exposure to certain wavelengths of light are used. The fluorescent label can be selected from the group consisting of rhodamines, cyanines, pyrenes and derivatives thereof. Preferred fluorescent fluorophores are based on a xanthene nucleus, which can readily undergo stacking to form dimers. Especially suitable are rhodamine fluorophores.
Suitably the reporter moiety comprises any usable fluorescent label. Fluorescent labels with environmentally sensitive fluorescence are most suitable. When a cysteine is the site of attachment, then the moiety needs thiol-reactivity for attachment. In other embodiments, an amine-sensitive label on a non-Cys amino acid may be employed.
In some embodiments, reporter moieties may be those that can exhibit molecular stacking, which will thus include aromatic rings. These include the rhodamine labels. In other embodiments labels which do not stack may be used, such as coumarin labels.
Dye stacking is a non-covalent interaction between two chromophores having planar aromatic rings, and it occurs when the rings are separated by a distance that is short enough to allow them to interact e.g. to form dimers or trimers. The detectable signal of the stacked molecules is different from that of the unstacked molecules (e.g. stacking can cause quenching of signals, and so stacked chromophores will typically show a decreased fluorescence signal intensity relative to the individual unstacked chromophores), and this difference can be used to detect the presence or absence of stacking. Stacked chromophores can have absorption spectra with (i) a characteristic decrease in the principal absorption peak as chromophore concentration increases and (ii) a characteristic shoulder peak ('band splitting').
For example, rhodamine chromophores can form dimers at high concentrations in solution. The dimer has a different absorbance spectrum from the monomer, and has little or no fluorescence in comparison with the monomer. Two rhodamine chromophores attached to suitable positions in the protein can form dimers, whose interaction is altered when ligand binds to the protein. The invention can spectroscopically detect the difference between the ATP-free and ATP-bound conformations of ATP binding molecule. Molecular stacking takes place through the physical interaction of ground states of the two moieties. Labels that can undergo molecular stacking are well known in the art. Stacking can occur between identical chromophores, and can also occur between different chromophores.
Suitably the reporter moiety is a dye. Suitably the reporter moiety is or comprises a fluorophore. Suitably said fluorophore is attached at a position on the polypeptide such that conformational change of the polypeptide upon ATP binding causes a corresponding change in fluorescence of said fluorophore.
We describe ATP sensor molecules with single labelling. Most suitably, when the ATP sensor is singly labelled a coumarin type dye is used as the label. We describe ATP sensors having double labelling. Double labelling means two dye molecules per sensor molecule. Suitably each of the two dye molecules is attached to a separate amino acid residue on the sensor molecule. For doubly labelled ATP sensors. Suitably the dyes are rhodamine type dyes. For dual labelling, any stacking rhodamine type dye is useful.
Cy dyes may be useful in the invention. However, these might require different attachment points from those taught for rhodamine attachment due to different dye molecule sizes. In other words, the dye molecules may need to be placed closer together or further apart in space than the corresponding rhodamines. In some embodiments, suitably Cy dyes are not used, the reason is that Cy dyes can suffer from the drawback of tending to provide high fluorescence for much of the time. This can make it more difficult to observe the fluorescence changes which are useful in the invention.
Acrylodan dyes may be useful in the invention.
Dual Labelling
More specifically, for the two fluorophore stacking quenching (i.e. dual labelling as used for rho-MatB with two rhodamines):
In principle any rhodamine dye that can be specifically linked to surface thiols could be used. This includes the 5- and 6-isomers of tetramethylrhodamine.
Rhodamine dyes are available in different isomers. For example, 5- tetramethylrhodamine (5-ATR) and/or 6-ATR may be used to label ATP sensors of the invention. It is possible to use a mixture of rhodamine isomers to label the sensor of the invention. This may result in a heterogeneous population of sensor molecules. For example, if a mixture of 5-ATR and 6-ATR dye is used to label the sensor protein, the following species will be generated -
• 5-ATR, 5-ATR
• 5-ATR, 6-ATR
• 6-ATR, 5-ATR
• 6-ATR, 6-ATR
The sensor of the invention may comprise any of these species of molecules. The sensor of the invention may comprise a mixture of more than one of these sensor molecules. The sensor of the invention may comprise a mixture of all four of these labelled sensor molecules.
Without wishing to be bound by theory, it may be technically possible to find a difference in the absorption/emission behaviour of sensors according to the invention having different isomers of dye attached. However, in practice the use of a mixture of sensors having alternate isomer dyes attached performs well in the assays of the invention. Single Labelling
For single labelled sensors of the invention, any suitable dye may be used, most suitably a dye which provides a coplanar alignment when attached to the sensor protein. Other conformations are possible, for example a twisted conformation, but those often tend to provide a lower fluorescence. A lower fluorescence may be usable but would need to be checked on a case by case basis.
For singly labelled sensors for the invention, any coumarin type dye is expected to provide good results.
In principle, any fluorophore known to have an environmentally sensitive fluorescence may be useful in the invention. In more detail, for single labelling, such as with a single environmentally sensitive dye, any coumarin may be used, including the iodoacetamide- and maleimide- linked diethylaminocoumarins (N-[2-(i-maleimidyl)ethyl]-7-diethylaminocoumarin-3- carboxamide and N-[2-(iodoacetamido)ethyl]-7-diethylaminocoumarin-3- carboxamide).
Other fluorophore types, known to have fluorescence intensity, depending on physical environment , such as interactions with protein surfaces, include
CPM (7-(diethylamino)-3-[4'-(i-maleimidyl)phenyl]-4-methylcoumarin)
Alexafluor 350 - C5 maleimide
Acrylodan (6-acryloyl-2-dimethylaminonaphthalene)
Badan (6-bromoacetyl-2-dimethylaminonaphthalene)
MIANS (2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid)
IAEDANS (5-[2-[(2-Iodo-i-oxoethyl)amino]ethylamino]-i-naphthalenesulfonic acid) Alexa Fluor 488 maleimide
Fluorescein-5-maleimide
Cy3-maleimide (i-(6-{[2-(2,5-dioxo-2,5-dihydro-iH-pyrrol-i-yl)ethyl]amino}-6- oxohexyl)-2-[(iE,3E)-3-(i-ethyl-3,3-dimethyl-5-sulfo-i,3-dihydro-2H-indol-2- ylidene)prop-i-enyl]-3,3-dimethyl-3H-indolium)
Pyrene-maleimide Conjugation Chem istry
Reporter moieties or labels such as fluorophores may be attached to the ATP binding molecule of the invention by any suitable means known in the art. Suitable amino acid residues may be engineered into the polypeptide. Although the invention is exemplified using cysteines such as cysteine substitutions, and attachment of reporters (e.g. fluorophores) such as via maleimide or iodoacetamide, it should be borne in mind that alternate technologies such as peptide ligation (e.g. chemical synthesis of protein or attachment of synthetic peptides to other polypeptides), and/or introduction of unnatural amino acids using mutated tRNAs and/or tRNA synthetases may be used. For example, amino acids having reactive azide groups may be introduced to take advantage of 'click chemistry' (or vice versa) or other conjugation techniques may be used. For example, lysine based unnatural amino acids may be introduced to achieve this goal (e.g. Nguyen et al 2009 (J Am Chem Soc. 2009 Jul i;i3i(25):8720-i); e.g. Lang et al 2012 Nature Chemistry 4, 298-304 (2012)).
Suitably the fluorophores are attached by conventional conjugation techniques such as covalent attachment via a cysteine residue in the polypeptide component of the ATP binding molecule.
The covalent attachment of extrinsic reporter moieties to proteins is well known. Different cysteine residues show different reactivities to labelling reagents, which can be assessed using DTNB (5,5'-dithio-bis(2-nitrobenzoic acid)). Reporter moieties can be attached via amines or carboxyl residues on amino acid side chains, but it is more suitable to use covalent linkage via thiol groups on a cysteine residue. Where more than one label is attached to a protein, these are suitably attached to separate amino acid residues. Where a cysteine residue has to be introduced, either by insertion or substitution, a number of factors should be considered. These are discussed in more detail herein. Exemplary sites for introduction of Cys residues and thus for label attachment are disclosed in detail herein. If attached chromophores are to interact, the residues must be selected such that they are in proximity to each other, and that the conformational change that occurs on ATP- binding affects one or both of the residues to cause a change in position or orientation or electronic environment of a label attached thereto. Exemplary pairs of attachment sites are set out above.
Exemplary attachment techniques are presented in the examples section.
ASSESSMENT OF FLUORESCENCE
A key concept of the invention is that the ATP binding molecules (sensor proteins) are configured so that they undergo a conformational change upon ATP binding. It is detection of this conformational change which allows the ATP binding status of the molecules of the invention to be determined. Clearly, where the technology permits, single molecule conformational changes may be determined. However, for most embodiments of the invention, determination of the conformational status of the ATP binding molecule is suitably assessed as a population effect. In other words, assessing the conformational change of an ATP binding molecule of the invention may be carried out by determining the conformational change of a population of ATP binding molecules of the invention. Considering those embodiments where the conformational change is read out or detected by monitoring changes in the behaviour of a reporter moiety coupled to the ATP binding molecule of the invention, this idea may be explained as follows. For the purposes of illustration, the reporter moiety will be considered to be a fluorophore. The fluorophore is attached to the ATP binding molecule of the invention. ATP binding leads to a conformational change of the ATP binding molecule. This conformational change can lead to a change in fluorescence. This change in fluorescence may be an increase or a decrease upon ATP binding depending on the particular labelling strategy used. For any given application having a fixed amount of sensor protein, the change in fluorescence will be consistently associated with the corresponding change in ATP binding. ATP binding is proportional to the concentration of ATP present in the sample being studied. Therefore, changes in ATP binding provide information about changes in the ATP concentration in the sample being studied. Thus, changes in fluorescence which are catalysed by conformational changes in the ATP binding molecule of the invention brought about by ATP binding directly provide information about the concentration of ATP in the sample being studied. For the great majority of applications or embodiments of the invention, conformational changes will be detected for a population of ATP binding molecules according to the invention. In practical terms, this means that a certain amount of the ATP binding molecule of the invention will be added to the sample being studied. The fluorescence of this population of ATP binding molecules of the invention will then be monitored. Thus, changes in the fluorescence of these molecules represents an indication of the ATP binding status of a proportion or a population of those molecules present in the sample. Thus, it will be clearly understood that the level of fluorescence varies directly with the amount of ATP binding of the ATP binding molecules of the invention. Thus, this will give the effect of an almost continuously variable level of fluorescence depending on the level of ATP binding (and thus the ATP concentration) within the sample. At one extreme, there will be no ATP binding to any of the ATP binding molecules of the invention. At the other extreme, all of the ATP binding molecules of the invention present in the sample will each be bound to ATP such that there is saturation and complete (or near complete) binding and therefore a ioo% signal. Thus, within these two extremes, the particular level of fluorescence is directly correlated to a particular concentration of ATP in the sample. Thus, in some embodiments of the invention, a standard curve may be constructed by measuring the fluorescence of a constant amount of the ATP binding molecule of the invention in the presence of differing known concentrations of ATP. This standard curve may then be used in order to read out or convert measured fluorescence values to absolute concentrations of ATP present in a sample.
In another embodiment, the readout of the invention may be advantageously calibrated by inclusion of samples having known ATP concentrations in the analysis being undertaken. In these embodiments, the samples containing known concentrations of ATP may be regarded as "internal controls". This permits accurate estimation of ATP concentrations in experimental settings where reference to a standard curve is less appropriate, for example in complex reaction mixtures in which other components might perturb the readouts, or might not have been present during the construction of a standard curve, thereby making such comparisons potentially inappropriate. It will be noted that some of the sensors provided herein show increasing fluorescence in the presence of ATP, and some show decreasing fluorescence in the presence of ATP. Either type of sensor is useful.
Advantageously, sensors showing increased fluorescence upon binding ATP are used. These provide the advantage of avoiding confounding factors which might otherwise reduce fluorescence, for example photo-bleaching or other degradation of the dye. Occasionally a "percentage change" is discussed in the context of the invention. For sensors which increase their fluorescence upon the ATP binding, the percentage change is the percentage increase in fluorescence from the unbound to the bound state. For those sensors which exhibit a fluorescence decrease upon ATP binding, the percentage change is calculated upwards i.e. taking the decreased fluorescence observed on binding and comparing that to the higher level of fluorescence observed in the absence of ATP gives a percentage increase. For example, a sensor whose fluorescence decreases from 1.0 to 0.6 upon ATP binding has a percentage fluorescence change of 67% (i.e. the difference in fluorescence intensity of 0.4 divided by the fluorescence on ATP binding of 0.6 equals 67% change).
Suitably sensors of the invention exhibit at least 50% fluorescence change upon ATP binding, more suitably 60%, more suitably 70%, more suitably 80%, more suitably 90%, more suitably 100% or even more (such as a multiple of fluorescence in the unbound state).
DEFINITIONS
The term 'comprises' (comprise, comprising) should be understood to have its normal meaning in the art, i.e. that the stated feature or group of features is included, but that the term does not exclude any other stated feature or group of features from also being present.
An ATP binding molecule is a molecule capable of binding ATP. Use of the term ATP binding molecule does not imply or require that ATP is present. ATP binding molecule means molecule capable of binding ATP.
Abbreviations:
Rhodopseudomonas palustris malonyl-coenzymeA synthetase (RpMatB) - Protein Data Bank (PDB) - 5-iodoacetamidotetramethylrhodamine (5-IATR) - 7-diethylamino-3- ((((2-maleimidyl)ethyl)amino)carbonyl)coumarin (MDCC) - 7-diethylamino-3-((((2- iodoacetomido)ethyl)amino)carbonyl)coumarin (IDCC)) - size-exclusion chromatography coupled to multi-angle laser light scattering (SEC-MALLS) - nicotinamide adenine dinucleotide (NADH) - deoxyadenosine triphosphate (dATP) - adenosine 5'(Y-thio)triphosphate (ATPyS) - adenosine 5'-( ,Y-imido)triphosphate (AMP-PNP)
SLU: 5'-0-[N-(DEHYDROLUCIFERYL)-SULFAMOYL] ADENOSINE ADVANTAGES
It is an advantage that the sensor of the invention is faster than any existing ATP sensor molecule such as those based on polypeptides.
An existing ATP sensor molecule known as "ATeam" has a 30 fold higher affinity for ATP than for ADP. The sensors of the invention advantageously have approximately 67 fold higher affinity for ATP than for ADP. This is a significant advantage offered by sensors of the invention.
In more detail, in assay scenarios in which ATP is measured, ADP will almost always be present. Therefore, this difference in affinity for ATP compared to ADP provides a much better sensor than prior art sensors. The practical advantage is that sensors can tolerate much higher concentrations of ADP whilst still performing well as ATP sensors.
The exemplary biosensors shown have the advantage of larger signal.
Suitably the ATP binding molecule of the invention is, and may be used as, a reagentless biosensor.
The invention advantageously provides a reagentless biosensor for ATP.
The invention advantageously provides a fluorescent reagentless biosensor for ATP.
CO-FACTORS
Divalent cation such as divalent metal ion is required for ATP binding to the sensors of the invention.
Suitably one or more of Cd2+ (Cadmium), Mn2+ (manganese) and/or Mg2+ (Magnesium) should be present together with the sensor of the invention in use. Most suitably the divalent cation is Mg2+ (Magnesium). The skilled person knows that cations are not provided in isolation, but are provided as a salt with corresponding anions in solution. A typical way of providing divalent cations such as Mg2+ is to add magnesium chloride (MgCl2) to the reaction mixture/buffer. However, any other acceptable salt of the above mentioned divalent metal ions may equally be used provided it does not compromise the action of the assay. This is easily tested as set out below in the example section whilst varying the divalent cation (i.e. the salt) which is incorporated into the assay.
In one embodiment the invention relates to a kit comprising an ATP sensor molecule as described above together with a source of divalent Magnesium ion (Mg2+). Suitably the source of Mg2+ is Magnesium Chloride. Most importantly, in the presence of divalent cation such as divalent metal ion e.g. Mg2+, ATP will be present as MgATP. Suitably Mg2+ is present in excess over ATP in the assay of the invention. Suitably Mg2+ is present at imM or more in the assay of the invention. Suitably ATP is present as MgATP in the assay of the invention. pH
The sensors of the invention have the advantage of being usable under a wide range of pH conditions. Suitably the pH of the assay is in the range 6.0 to 9.0. More suitably the pH of the assay is in the range 7.0 to 7.5.
It is an advantage of the invention that the sensor molecules do not react to nucleotides other than ATP, nor to ATP analogues. In other words, it does not matter if these chemical entities are also present in the assay mixture, the sensor has the advantage of only reacting to ATP and therefore the presence of these other molecular species does not perturb the assay of ATP concentration according to the invention.
FURTHER APPLICATIONS AND EMBODIMENTS
The invention provides a fluorescent, reagentless biosensor for ATP, suitably based on or derived from malonyl-coenzyme A synthetase.
Additional mutations may be made to the sensors as described above to acquire further advantages.
Examples of mutations which may be made are shown in Table C (see example 13). Table C shows the characteristics bestowed on the sensor by making of the mutations as shown. Therefore, depending on the application(s) of the biosensor intended by the skilled worker, mutations from Table C should be chosen accordingly, or not made, as desired. In Table C, 'Parent' means exemplary Rho-MatB; however, the mutations in Table C are equally applicable to any other biosensor disclosed herein without requiring the specific set of mutations of Rho-MatB - these additional mutations of Table C are merely exemplified in the Rho-MatB background to illustrate the advantages and help understand the effects of the mutations which are described individually and may be applied to biosensors of the invention individually or in combination. Suitably mutations disclosed in Table C are applied individually. Suitably the invention provides a biosensor as described above further comprising one further mutation selected from those disclosed in Table C.
In more detail, especially useful mutations are discussed more fully below.
Mutating T167 provides the advantage of increased Kd for ATP whilst retaining excellent fluorescence ratio (F+/F-). This has the further technical benefit of widening the range of ATP concentrations that can be measured by the biosensor.
Suitably T167 is mutated to a small amino acid.
Suitably T167 is mutated to Alanine (A), Serine (S) or Glycine (G).
Glycine can make the chain flexible which may or may not be desired. Thus suitably T167 is mutated to Alanine (A) or Serine (S), avoiding flexibility effect(s) of Glycine. Thus suitably said polypeptide comprises an amino acid other than T at the position corresponding to 167 of SEQ ID NO: 1. Thus suitably said polypeptide comprises Alanine (A), Serine (S) or Glycine (G) at the position corresponding to 167 of SEQ ID NO: 1. More suitably said polypeptide comprises Alanine (A) or Serine (S) at the position corresponding to 167 of SEQ ID NO: 1. More suitably said polypeptide comprises Serine (S) at the position corresponding to 167 of SEQ ID NO: 1, which has the advantage of enhanced fluorescence ratio and enhanced Kd compared to Rho- MatB. Most suitably said polypeptide comprises Alanine (A) at the position corresponding to 167 of SEQ ID NO: 1, which has the advantage of greatly enhanced Kd whilst retaining the same advantageous fluorescence ratio as Rho-MatB.
Sl70
Mutating S170 provides the advantage of increased fluorescence ratio (F+/F-) whilst retaining excellent Kd for ATP. This has the further technical benefit of a decreased ADP affinity, thereby increasing selectivity of ATP over ADP to >200 fold. This is especially advantageous in measuring ATP when ADP is also present.
Suitably S170 is mutated to a small amino acid.
Suitably S170 is mutated to Alanine (A) or Glycine (G).
Glycine can make the chain flexible which may or may not be desired. Thus suitably S170 is mutated to Alanine (A), avoiding flexibility effect(s) of Glycine.
Thus suitably said polypeptide comprises an amino acid other than S at the position corresponding to 170 of SEQ ID NO: 1. Thus suitably said polypeptide comprises Alanine (A), or Glycine (G) at the position corresponding to 170 of SEQ ID NO: 1. More suitably said polypeptide comprises Alanine (A) at the position corresponding to 170 of SEQ ID NO: 1. Most suitably said polypeptide comprises Alanine (A) at the position corresponding to 170 of SEQ ID NO: 1, which has the advantage of greatly increased fluorescence ratio (F+/F-) compared to Rho-MatB. T303
Mutating T303 provides the advantage of increased Kd for ATP whilst retaining excellent fluorescence ratio (F+/F-). This has the further technical benefit of widening the range of ATP concentrations that can be measured by the biosensor.
Suitably T303 is mutated to a small amino acid.
Suitably T303 is mutated to Alanine (A), Serine (S) or Glycine (G).
Glycine can make the chain flexible which may or may not be desired. Thus suitably T303 is mutated to Alanine (A) or Serine (S), avoiding flexibility effect(s) of Glycine. Thus suitably said polypeptide comprises an amino acid other than T at the position corresponding to 303 of SEQ ID NO: 1. Thus suitably said polypeptide comprises Alanine (A), Serine (S) or Glycine (G) at the position corresponding to 303 of SEQ ID NO: 1. More suitably said polypeptide comprises Alanine (A) or Serine (S) at the position corresponding to 303 of SEQ ID NO: 1. More suitably said polypeptide comprises Serine (S) at the position corresponding to 303 of SEQ ID NO: 1, which has the advantage of enhanced Kd compared to Rho-MatB. Most suitably said polypeptide comprises Alanine (A) at the position corresponding to 303 of SEQ ID NO: 1, which has the advantage of enhanced Kd whilst also retaining an advantageous high fluorescence ratio comparable to that of Rho-MatB.
Suitably positions corresponding to T167 and T303 of SEQ ID NO: 1 are not both mutated in the same sensor molecule. Without wishing to be bound by theory, it is possible that double mutation of positions 167 and 303 may weaken ATP binding in such a double mutant. Thus suitably if a position corresponding to T167 is mutated, then the position corresponding to T303 is wild type, suitably T. Thus suitably if a position corresponding to T303 is mutated, then the position corresponding to T167 is wild type, suitably T. Most suitably the additional mutations described are made in conjunction with the mutations of RhoMatB such as shown in SEQ ID NO: 6. Suitably the dyes and any other additions to the sensor molecule are as for RhoMatB.
INDUSTRIAL APPLICATION
The invention finds use as a reagent in a research setting.
The invention finds application in the assessment of food contamination, such as bacterial contamination.
The invention finds application in drug screening methodology.
The invention finds application in any setting in which it is desired to assay ATP. The invention finds application in the assessment of surface contamination such as in a clinical/hospital/medical environment.
The invention finds application in the assessment of surface contamination such as in food preparation areas.
Further particular and preferred aspects are set out in the accompanying independent and dependent claims. Features of the dependent claims may be combined with features of the independent claims as appropriate, and in combinations other than those explicitly set out in the claims.
Where an apparatus feature is described as being operable to provide a function, it will be appreciated that this includes an apparatus feature which provides that function or which is adapted or configured to provide that function. BRIEF DESCRIPTION OF THE DRAWINGS
Embodiments of the present invention will now be described further, with reference to the accompanying drawings, in which: Figure 1: C-terminal domain rotation upon MgATP binding and position of mutations in RpMatB. (A) Shown is a cartoon representation of RpMatB. The N- terminal domain (amino acids 1-399) in the apo conformation (PDB 4FUQ (Crosby, Rank et al. 2012)) is coloured grey. The C-terminal domain (amino acids 400-503) in the apo conformation is shown in pink. The C-terminal domain in the MgATP-bound conformation (PDB 4FUT (Crosby, Rank et al. 2012), after superimposing the N- terminal domain of 4FUT on the N-terminal domain of 4FUQ) is shown in green. ATP (in ball and stick conformation and coloured by CPK convention) and Mg2+ (depicted as an orange sphere) are shown in the active site. The C-terminal domain rotation upon MgATP-binding is clearly visible. The positions of mutations are shown as spheres and labeled: C106A in the N-terminal domain (yellow), K488A in the C-terminal domain (red), R286C in the N-terminal domain and Q457C in the C-terminal domain (both blue). (B) As described above but the C-terminal domain in the malonyl-coenzymeA and AMP bound conformation of Streptomyces coelicolor MatB (PDB 3NYR (Hughes and Keatinge-Clay 2011), after superimposing the N-terminal domain of 3NYR on the N-terminal domain of 4FUQ) is shown in blue. Malonyl-coenzymeA and AMP (in ball and stick conformation and coloured by CPK convention) are shown in the active site.
Figure 2: Characterization of the fluorescence and absorbance spectra of Rho- MatB. (A) Fluorescence excitation and emission spectra of 1 μΜ Rho-MatB in 50 mM Hepes pH 7.0, 100 mM NaCl, 10 mM MgCl2 and 0.3 mg ml"1 bovine serum albumin in the absence and the presence of 175 μΜ ATP. Excitation was at 553 nm for the emission spectra. Emission was measured at 575 for the excitation spectra. Data were corrected for dilution. (B) Absorbance spectra of 1 μΜ Rho-MatB in 50 mM Hepes pH 7.0, 100 mM NaCl, 10 mM MgCl2, 0.3 mg ml"1 bovine serum albumin in the absence and presence of 119 μΜ ATP. Data were corrected for dilution.
Figure 3: Characterization of nucleotide binding affinity to Rho-MatB. (A)
Titration of ATP (open circles), ADP (solid circles), dATP (open triangles), ATPyS (solid triangles) and AMP-PNP (open squares) to 0.5 μΜ Rho-MatB in 50 mM Hepes pH 7.0, 100 mM NaCl, 10 mM MgCl2, 0.3 mg ml"1 bovine serum albumin. Aliquots of ligand were added and the fluorescence intensity was measured at 571 nm (exciting at 553 nm) at 20 °C. The data were corrected for dilution and normalized to 1 for the fluorescence intensity of Rho-MatB in the absence of ligand. The dissociation constants were obtained using a quadratic binding curve (see Materials and methods). Shown here is one representative experiment. The dissociation constants listed in Table 1 are the average of at least three repeat experiments. (B) 1 uM Rho-MatB in the absence (open circles) and presence of 100 μΜ ADP (solid circles), 100 μΜ dATP (open triangles), 100 μΜ ATPyS (solid triangles) or 100 μΜ AMP-PNP (open squares) in 50 mM Hepes pH 7.0, 100 mM NaCl, 10 mM MgCl2 and 0.3 mg ml"1 bovine serum albumin was titrated with an ATP / ADP, dATP, ATPyS or AMP-PNP / Rho-MatB solution with ADP, dATP, ATPyS or AMP-PNP and Rho-MatB of the same concentration as ADP, dATP, ATPyS or AMP-PNP and Rho-MatB in the cuvette. That means that [ADP], [dATP], [ATPyS] or [AMP-PNP] and [Rho-MatB] were held constant. The fluorescence intensity was measured at 571 nm (exciting at 553 nm) at 20 °C. The data were normalized to 1 for the fluorescence intensity of Rho-MatB in the absence of ATP. The dissociation constants for ATP were obtained using a quadratic binding curve (see Materials and methods). Shown here is one representative experiment. The dissociation constants listed in Table 1 are the average of two repeat experiments. (C) 2.5 μΜ Rho- MatB in the absence (open circles) and presence of 10 μΜ ADP (solid circles), 50 μΜ ADP (solid triangles) or 100 μΜ ADP (solid squares) in 50 mM Hepes pH 7.0, 100 mM NaCl, 10 mM MgCl2 and 0.3 mg ml"1 bovine serum albumin was titrated with an ATP / Rho-MatB solution with ATP and Rho-MatB of the same concentration as ADP and Rho-MatB in the cuvette. That means that the total nucleotide concentration ([ATP] + [ADP]) and [Rho-MatB] was held constant and only the proportion of ATP was changed. The fluorescence intensity was measured at 571 nm (exciting at 553 nm) at 20 °C. The data were normalized to 1 for the fluorescence intensity of Rho-MatB in the absence of ATP. The fluorescence response could be fitted to a line up to 6 μΜ ATP with - for the experiment shown - slopes of 0.178 ± 0.010, 0.180 ± 0.004, 0.153 ± 0.005 and 0.124 ± 0.004 μΜ"1 at 0, 10, 50 and 100 μΜ ADP, respectively.
Figure 4: Association and dissociation kinetics of Rho-MatB and ATP. (A, B)
Association kinetics were measured under pseudo-first order conditions with respect to ATP by mixing 0.25 μΜ Rho-MatB with different concentrations of ATP at 25 °C in 50 mM Hepes pH 7.0, 100 mM NaCl, 10 mM MgCl2, 0.3 mg ml"1 bovine serum albumin using a stopped-flow apparatus. Two time windows were used to in order to accurately measure the fast phase and the slow phase. A representative set of fluorescence traces is shown in black and the labels are the micromolar ATP concentrations. Data were fitted using the Kinetic Studio software. The fitted traces are shown in red. The short time scale fluorescence signal observed after mixing Rho-MatB and ATP was well fitted using a single exponential with a linear term. The long time scale fluorescence signal fitted to a double exponential function using a fixed rate constant for the fast phase determined previously from the short time scale traces. The average rate constant determined for the slow phase is 0.88 ± 0.13 s"1. The observed rate constants for the fast phase (k0bS,fast) are plotted against ATP concentration (C). Linear regression gave an association rate constant of 1.83 ± 0.02 μΜ"1 s"1 and the intercept was 8.15 ± 0.17 s"1. (D) Association kinetics were measured under pseudo-first order conditions with a 5 or 10-fold excess of Rho-MatB over ATP (0.25 μΜ ATP and 1.25 μΜ Rho-MatB; 0.50 μΜ ATP and 2.50 μΜ Rho-MatB; 0.375 μΜ ATP and 3.75 μΜ Rho-MatB; 0.500 μΜ ATP and 5.00 μΜ Rho-MatB; 0.625 μΜ ATP and 6.25 μΜ Rho-MatB; 0.750 μΜ ATP and 7.50 μΜ Rho-MatB or 1.00 μΜ ATP and 10.0 μΜ Rho-MatB). A representative set of fluorescence traces, recorded at 25 °C in 50 mM Hepes pH 7.0, 100 mM NaCl, 10 mM MgCl2, 0.3 mg ml"1 bovine serum albumin using a stopped-flow apparatus, is shown in black. Each trace is normalized to 100% for the initial intensity but offset by 2% from each other for clarity. The concentrations mentioned in the graph are those of Rho-MatB in the mixing chamber. Data were fitted with a single exponential using the Kinetic Studio software. The fitted traces are shown in red. The observed rate constants are plotted against Rho-MatB concentration (E). Linear regression gave an association rate constant of 0.72 ± 0.03 μΜ"1 s"1 and the intercept was 7.95 ± 0.18 s"1. (F) To determine the dissociation rate constants, we mixed the preformed ATP (5 μΜ) and Rho-MatB (0.25 μΜ) complex with 25 μΜ (solid line) or 50 μΜ (dashed line) His6- RpMatB C106A/K488A at 25 °C in 50 mM Hepes pH 7.0, 100 mM NaCl, 10 mM MgCl2, 0.3 mg ml"1 bovine serum albumin in a stopped-flow apparatus. Representative time courses are shown. The kinetics of the fluorescence change are limited by the ATP dissociation, as shown by varying the concentration of His6-RpMatB C106A/K488A. Data were fitted with a double exponential using the Kinetic Studio software. The average rate constants determined are 6.94 ± 0.28 s"1 (-85 %) and 1.50 ± 0.07 s"1 (-15 %). The percentages in parentheses are the relative intensities of the fast and slow phases for double-exponential fits, these were approximately constant as the concentration varied. Figure 5: (A) Conformational selection model. Rho-MatB 1 and Rho-MatB2 correspond to two different conformational states of Rho-MatB. Only Rho-MatB2 can bind ATP to form ATP RhoMatB2. (B) Induced fit model. Rho-MatB binds ATP to form ATP RhoMatB. ATP RhoMatB* corresponds to ATP-bound Rho-MatB in a different conformational state.
Figure 6: The production of ATP by pyruvate kinase as monitored by Rho-MatB.
(A) The calibration curve to define the relationship between fluorescence intensity and ATP concentration. The fluorescence intensity of a solution containing 2.5 μΜ RhoMatB in 50 mM Tris HC1 pH 7.5, 100 mM NaCl, 10 mM MgCl2, 100 mM KC1, 0.3 mg ml"1 bovine serum albumin, 250 μΜ ADP, 2.5 mM phosphoenolpyruvate and ATP was measured in a microplate reader. A linear relationship between fluorescence intensity and ATP concentration at these conditions was observed up to 5 μΜ ATP. The slope obtained by linear regression analysis is shown. (B) Time courses of fluorescence change upon ATP production by pyruvate kinase at different phosphoenolpyruvate concentrations as monitored by Rho-MatB using a microplate reader. Reaction mixtures contained 50 mM Tris HC1 pH 7.5, 100 mM NaCl, 10 mM MgCl2, 100 mM KC1, 0.3 mg ml"1 bovine serum albumin, 250 μΜ ADP, 2.5 μΜ Rho-MatB and various phosphoenolpyruvate concentrations. All reactions were started by the addition of pyruvate kinase (0.025 U ml"1), and the change in fluorescence was monitored for several minutes at room temperature (-20 °C). The initial rates (vi) were determined by linear regression (from 200 to 400 s) using the slope obtained from the calibration curve and were plotted versus phosphoenolpyruvate concentration (C). The parameters Km and Vmax were obtained from a curve fit according to the Michaelis-Menten equation and are - for the experiment shown - 75.5 ± 3.4 μΜ and 0.019 ± 0.002 μΜ s"1, respectively. (D) Time courses of ATP production by pyruvate kinase as monitored by Rho-MatB in a stopped-flow apparatus. Reaction mixtures contained 50 mM Tris HC1 pH 7.5, 100 mM NaCl, 10 mM MgCl2, 100 mM KC1, 0.3 mg ml"1 bovine serum albumin, 250 μΜ ADP, 100 μΜ phosphoenolpyruvate and 2.5 μΜ Rho-MatB. All reactions were started by the addition of pyruvate kinase (0.025, 0.50, 0.75, 1.0, 2.0, 4.0, 5.0 or 6.0 U ml"1), and the change in fluorescence was monitored for several seconds at 25 °C. ATP concentrations were calculated from the fluorescence signal using the calibration method. Briefly, the signal was calibrated by consecutively introducing 2.5 μΜ Rho-MatB alone in 50 mM Tris HC1 pH 7.5, 100 mM NaCl, 10 mM MgCl2, 100 mM KC1, 0.3 mg ml"1 bovine serum albumin, 250 μΜ ADP, 100 μΜ phosphoenolpyruvate; then the same solution containing 0.5, 1.0, 2.0, 3.0, 4.0 or 5.0 μΜ ATP. At each stage the fluorescence was measured. A representative set of traces at different pyruvate kinase concentrations is shown. Traces are offset by 0.20 μΜ ATP from each other at zero time for clarity. The initial rates (vi) were determined by linear regression and were plotted versus pyruvate kinase concentration (E).
Figure 7 (Supplemental figure 1): Chromatogram of the purification of Rho-MatB via ion exchange chromatography. Detection was performed via A28onm- The conductivity (red) is shown on the secondary vertical axis. The conductivity signal gives an indication of the applied gradient. See Materials and methods for further details.
We observed that the protein eluted in two peaks. Overall, our data suggested that the protein was in equilibrium between two "states". For instance, when one peak was rerun using the same conditions, we could see the same elution profile again (data not shown). Also, there is no difference in the fluorescence change upon ATP -binding and the affinity for ATP for both peaks (data not shown).
Figure 8 (Supplemental figure 2): Characterization of the fluorescence spectra of Rho-MatB. (A) Fluorescence emission spectra of 1 μΜ Rho-MatB in 50 mM Hepes pH 7.0, 100 mM NaCl, 10 mM MgCl2 and 0.3 mg ml"1 bovine serum albumin (black line) supplemented with 1 mM malonate (red line), 0.25 mM coenzymeA (blue line), 1 mM malonate and 0.25 mM coenzymeA (green line) or 1 mM malonate, 0.25 mM coenzymeA and 1 mM ATP (orange line). Data were corrected for dilution. (B) Fluorescence emission spectra of 1 μΜ Rho-MatB in 50 mM Hepes pH 7.0, 100 mM NaCl, 0.5 mM EDTA and 0.3 mg ml"1 bovine serum albumin (black line) supplemented with 250 μΜ ATP (red line) or 250 μΜ ATP and 10 mM MgCl2 (blue line). Data were corrected for dilution.
The fluorescence emission spectra were measured by exciting protein solutions at 553 nm. Figure 9 (Supplemental figure 3): Simulations of ATP-binding to Rho-MatB. (A)
Simulation using the conformational selection model (Figure 5A). Simulated time traces shown are, from bottom to top, 0.25 μΜ ATP : 1.25 μΜ Rho-MatB; 0.375 μΜ ATP: 3.75 μΜ Rho-MatB; 0.625 μΜ ATP: 6.25 uM Rho-MatB and 1.00 μΜ ATP: 10.0 μΜ Rho-MatB. The simulated time traces are normalized to 100% for the initial signal but offset by 2% from each other for clarity. The observed fluorescence signal was generated by combined fluorescence from all species using the following formula: a χ ([Rho-MatB 1] + [Rho-MatB2]) + b χ [ATP RhoMatB2] with a = 1 and b = 3.7. Rho- MatB 1 and Rho-MatB2 concentrations were both set at 50 % of the total [Rho-MatB]. Rate constants were k+i = 0.88 s"1, k.i = 0.88 s"1, k+2 = 1.83 μΜ"1 s"1 and k-2 = 8.15 s"1. (B) Simulation using the induced fit model (Figure 5B). Simulated time traces shown are, from bottom to top, 0.25 μΜ ATP : 1.25 μΜ Rho-MatB; 0.375 uM ATP: 3.75 μΜ Rho-MatB; 0.625 μΜ ATP: 6.25 μΜ Rho-MatB and 1.00 μΜ ATP: 10.0 μΜ Rho- MatB. The simulated time traces are normalized to 100% for the initial signal but offset by 2% from each other for clarity. The observed fluorescence signal is generated by combined fluorescence from all species using the following formula: a χ [Rho-MatB] + b x [ATP RhoMatB] + c χ [ATP · RhoMatB * ] with a = 1, b = 3 and c = 3.7. Rate constants were k+1 = 1.83 μΜ"1 s"1, k.i = 8.15 s"1, k+2 = 0.88 s"1 and k-2 = 0 s"1. Figure 10 (Supplemental figure 4): Rho-MatB activity assay. We measured the time course and extent of NADH oxidation in the presence of Rho-MatB to determine the residual activity of Rho-MatB. Reaction mixtures (200 μΐ) contained 50 mM Hepes buffer pH 7.5, 25 mM NaCl, 25 mM KC1, 0.3 mg ml"1 bovine serum albumin, 0.5 mM ATP, 0.5 mM coenzymeA, 10 mM MgCl2, 3 mM phosphoenolpyruvate, 0.2 mM NADH, 0.01 U μΐ"1 pyruvate kinase, 0.05 U μΐ"1 adenylate kinase, 0.015 U μΐ"1 lactate dehydrogenase and 2 mM malonate. The reaction was started by the addition of 0.03 μΜ Rho-MatB and the change in A340nm was monitored in time. No activity was observed. As a positive control, 0.03 μΜ (5-ATR)2-His6-RpMatB C106A/R286C/Q457C was added to the reaction mixture. The specific activity for (5- ATR)2-His6-RpMatB C106A/R286C/Q457C was calculated using 8340nm,NADH and was 37 ± 4 μιηοΐ AMP min"1 mg"1.
Figure 11 (Supplemental figure 5): The production of ATP by pyruvate kinase as monitored by a coupled-enzyme assay. (A) Reaction mixtures (200 μΐ) contained 50 mM Tris HCl pH 7.5, 100 mM NaCl, 10 mM MgCl2, 100 mM KC1, 0.3 mg ml"1 bovine serum albumin, 250 μΜ ADP, 200 μΜ NADH, 5 U ml"1 lactate dyhydrogenase and various concentrations of phosphoenolpyruvate. All reactions were started by the addition of pyruvate kinase (0.10 U ml"1) and the change in A340nm was monitored for several minutes at room temperature (~ 20 °C) using a microplate reader. The initial rates (vi) were determined by linear regression (from 200 to 700 s) using 8340nm,NADH and were plotted versus phosphoenolpyruvate concentration. (B) The parameters Km and Vmax were obtained from a curve fit according to the Michaelis-Menten equation and are - for the experiment shown - 173 ± 19 μΜ and 0.063 ± 0.002 μΜ s"1, respectively. Figure 12 (Supplemental figure 6): The quaternary structures of His6-RpMatB C106A/R286C/Q457C/K488A and Rho-MatB were analyzed via SEC-MALLS.
About 15 μΜ Rho-MatB (blue) and His6-RpMatB C106A/R286C/Q457C/K488A (red) were analyzed via SEC -MALLS at room temperature at a flow rate of 0.5 ml min"1 in the absence of ATP (30 mM Tris HCl pH 7.5, 100 mM NaCl, 3 mM NaN3, graph on the left) and in the presence of ATP and Mg2+ (30 mM Tris HCl pH 7.5, 100 mM NaCl, 5 mM MgCl2, 50 μΜ ATP, 3 mM NaN3, graph on the right). The differential refractive index and the molar mass distributions, determined throughout the elution of each peak, were plotted against time.
Briefly, in the absence of ATP or Mg2+, we observe a broad peak with fronting corresponding to a molar mass ranging from -60 to 80 kDa for His6-RpMatB C106A/R286C/Q457C/K488A and a molar mass ranging from -60 to 90 kDa for RhoMatB. At higher concentrations of His6-RpMatB C106A/R286C/Q457C/K488A, the equilibrium shifts toward the 60 kDa form (data not shown). Addition of 50 μΜ ATP in the presence of 5 mM Mg2+ shifts the equilibrium towards the 60 kDa species for the unlabeled protein. However, no clear shift is observed for Rho-MatB, i.e. its molar mass ranges from -60 to 80 kDa.
To conclude, we observe an equilibrium between two species. This can be monomer and dimer protein. However, it is also possible that the protein exists as two different conformations in solution. This equilibrium is not only concentration-dependent but also ligand-dependent.
Figure 13 (Table 1): Fluorescence changes (F+/F.) and dissociation constants (Kd) for binding of ATP and other ligands to Rho-MatB. The fluorescence change upon ligand binding and the equilibrium dissociation constants were obtained from fluorescence titrations as described in Figure 3A and Figure 3B.
Figure 14 (Supplemental table 1): Fluorescence changes (F+/F.) and dissociation constants (Kd) for binding of ATP and ADP to diethylaminocoumarin (MDCC and IDCC) and tetramethylrhodamine (5-ATR and 6-ATR) labeled RpMatB cysteine mutants. Data are from a survey without complete optimization of the labeling and purification for each mutant. The fluorescence changes upon ligand binding were obtained from fluorescence emission spectra at 20 °C in 50 mM Tris HC1 pH 7.5, 100 mM NaCl, 10 mM MgCl2, 0.3 mg / ml bovine serum albumin using 1 μΜ protein and excess ATP or ADP. The equilibrium dissociation constants were obtained from fluorescence titrations at 20 °C in 50 mM Tris HC1 pH 7.5, 100 mM NaCl, 10 mM MgCl2, 0.3 mg / ml bovine serum albumin using 0.5 μΜ protein and various concentrations of ATP or ADP.
Figure 15 (Supplemental table 2): Fluorescence changes (F+/F.) and dissociation constants (Kd) for binding of ATP to Rho-MatB in different buffer conditions. The fluorescence changes upon ATP binding were obtained from fluorescence emission spectra at 20 °C in the buffer mentioned using 1 μΜ protein and excess ATP in the presence of 10 mM MgCl2 and 0.3 mg ml"1 bovine serum albumin. The equilibrium dissociation constants were obtained from fluorescence titrations at 20 °C in the buffer mentioned using 0.5 μΜ protein and various concentrations of ATP.
Figure 16 (Supplemental Table 3): Structure comparison between apo and holo (ATP (analogue) -bound) ANL superfamily proteins.
To investigate the degree of the conformational change after ATP-binding within the ANL superfamily, we collected 5 apo-holo protein pairs (based on (Gulick 2009), including a more up-to-date table on
Figure imgf000048_0001
The structural similarities/differences between apo and holo structures were investigated using the root mean square deviation (RMSD) of the alpha carbon atoms of aligned residues using SuperPose (Maiti, Van Domselaar et al. 2004). The table shows that not only RpMatB but also other ANL superfamily members undergo a conformational change after ATP (analogue) binding.
We also analyzed the relative interdomain orientations through manual inspection of the placement of the C-terminal domain with respect to its N-terminal domain after superposing the N-terminal domain. For the holo molecules, we found 9 out of 1 1 structures for which the interdomain geometry is conserved. On the contrary, the superposition of the apo molecules showed large variations in the positioning of the C- terminal domain relative to the N-terminal domain.
(%) In total, we analyzed 11 holo molecules and 25 apo molecules. We found that the C-terminal domain was not present in 11 out of 25 analyzed apo crystal structures. It was either unresolved hence assumed to be flexible (like PDB 3IVR and PDB 4LGC) or not included in the purified protein (like PDB 3T5B and PDB 3WV4). For simplicity, only the 5 apo-holo pairs are shown in this table.
(+) Ratio of the average B-factor of the C-terminal domain using all atoms and the average B-factor of the N-terminal domain using all atoms.
(*) This crystal structure contained multiple chains that had a different conformation. Chains representing both conformations were used in the analysis.
($) Only partially resolved C-terminal domain.
(#) This crystal structure contained multiple chains but all had the same conformation. Only one was used in the analysis.
References to publications can be found using the links at the Protein Data Bank.
Figure 17 shows an exemplary sequence of the invention referred to as RpMatB, which is annotated to show examples of substitutions and/ or additions useful in sensors of the invention (SEQ ID NO: 7).
Figure 18 shows summary of the invention.
Figure 19 shows structural comparison of the proteins in Table B. Location of amino acids closest to those used in RpMatB as cysteine points of attachment.
Figure 20 shows C-terminal domain rotation upon MgATP binding and position of mutations in RpMatB. Figure 21 shows Crystal structure of RpMatB in the MgATP bound conform ation, showing conserved m otifs and position of binding site m utations.
(A) Protein structure showing the conserved core motifs and MgATP (Crosby, Rank et al. 2012). The highly conserved core motifs (see also Table Si) are shown: structural motifs are coloured green, binding site motifs are pink. (B) ATP binding site, showing the position of the three mutations.
Figure 22 shows Fluorescence excitation and em ission spectra of variants of Rho-MatB. (A) l μΜ Rho-MatB T167A with and without 5 niM ATP; (B) 1 μΜ Rho-MatB T303A with and without 3 mM ATP; (C) 1 μΜ Rho-MatB S170A with and without 0.5 mM ATP. These ATP concentrations were saturating for the variant. Solutions were in 50 mM Hepes pH 7.0, 100 mM NaCl, 10 mM MgCl2, 0.3 mg ml 1 bovine serum albumin at 20 °C.
Figure 23 shows Nucleotide affinity to variants of Rho-MatB.
Titration of ATP (circles) and ADP (triangles) (A) 0.5 μΜ Rho-MatB T167A, (B) 0.5 μΜ Rho-MatB T303A; (C) 0.5 μΜ Rho-MatB S170A. Measurements were in 50 mM Hepes pH 7.0, 100 mM NaCl, 10 mM MgCl2, 0.3 mg ml 1 bovine serum albumin at 20 °C. The dissociation constants and fluorescence ratios were obtained using a quadratic binding equation (see Methods) and are listed in Table D. The drop lines are to indicate the dissociation constants. Figure 24 shows The effect of ADP on ATP binding.
Calibrations were determined by measuring the fluorescence as a function of ATP with different amounts of ADP present, but with the total nucleotide concentration (ADP + ATP) constant. (A) 1 μΜ Rho-MatB T167A; (B) 1 μΜ Rho-MatB T303A; (C) 1 μΜ Rho- MatB S170A. The total nucleotide concentration is shown in micromolar. For Rho- MatB S170A, 500 μΜ ADP was added at each ATP concentration. Solution conditions were as in Figure 23. The data were linear fit to demonstrate approximate linear dependence over the range measured.
Figure 25 shows Association kinetics of ATP binding to Rho-MatB variants Fluorescence time courses were measured by rapidly mixing different concentrations of ATP with 0.25 μΜ Rho-MatB with a large excess of ATP (micromolar concentration shown) in 50 mM Hepes pH 7.0, 100 mM NaCl, 10 mM MgCl2, 0.3 mg ml 1 bovine serum albumin at 20 °C. Time courses are for two different time scales were obtained to show fast and slow phases. Slow phases are shown in Figure 28 (Figure S3). Note that the dead time of the stopped-flow instrument is ~2 ms, so that the traces of the fast phase only record changes from that time. (A) Example traces for Rho-MatB T167A, T303A and S170A variants. (B) The fast phases were fit to single exponentials to give rate constants (&0bs), increasing linearly with ATP concentration. A simple binding model for pseudo-first order conditions gives k0bs = &+[ATP] + k- the slope (second order rate constant for association, k+) and intercept (dissociation rate constant, k-) are shown in Table D. Figure 26 (Figure SI) shows core m otifs sequences Sequence logos were created for ANL superfamily proteins, using WebLogo 3.4
Figure imgf000051_0001
Sequence conservation is indicated as the total height of each stack (measured in bits), while the relative height of bases in a stack reflects base frequencies at that position. The numbers correspond to the alignment position. The colour scheme is based on hydrophobicity: R, K, D, E, N, Q are blue; S, G, H, T, A, P are green; Y, V, M, C, L, F, I, W are black. The motifs shown are ones in which mutations were prepared: the sequence of RpMatB is also shown for each. Figure 27 (Figure S2) shows Absorbance spectra of variants of Rho -MatB with and without ATP (A) 1 μΜ Rho-MatB T167A with and without 5 mM ATP; (B) 1 μΜ Rho-MatB T303A with and without 3 mM ATP; (C) 1 μΜ Rho-MatB S170A with and without 0.5 mM ATP. These ATP concentrations were saturating for the variant. Solutions were in 50 mM Hepes pH 7.0, 100 mM NaCl, 10 mM MgCl2, 0.3 mg ml 1 bovine serum albumin at 20 °C.
Figure 28 (Figure S3) shows Association kinetics of variants of Rho -MatB with excess ATP Example time courses were obtained as in Figure 25 at various ATP concentrations, shown in micromolar. Rho-MatB T167A, T303A and S170A variants. While Figure 25 shows the fast phases of each time course, the equivalent slow phase are shown here. These were fit to single exponentials, whose rate constants varied little with ATP concentration. The average rate constants, measuring a conformation change as described in the main text, are in Table D. EXAMPLES
Experim ental procedures
Selection of protein scaffold
All available structures of proteins expressed in E.coli with either ATP or an ATP analogue bound were extracted from the Protein Data Bank (PDB) (Berman, Westbrook et al, 2000). To obtain the corresponding ligand-free structure for each ATP or ATP analogue -bound structure, the sequences of all available PDB structures of proteins expressed in E.coli. were compared with the ATP and ATP analogue -bound sequences using CD-HIT2D (Huang, Niu et al. 20.10). This program identifies the sequences in one database that are similar to the other database. Sequences with 90% sequence identity were considered to be the same protein and allowed ligand-bound and ligand-free structures to be compared using PyMOL (The PyMOL Molecular Graphics System, Version 1.3, Schrodinger, LLC).
Plasmids
Plasmid pRpMatE>39 (plasmid pTEVs containing the coding sequence of RpMatB with the point mutation K488A and an N-terminal His6-tag) was provided by J.C. Escalante- Semerena (Co2.^ ...EanJ ...et...a]....20i2). The QuikChange site-directed mutagenesis protocol and the QuikChange Lightning Multi site-directed mutagenesis kit (Stratagene) were used for single-site or multi-site mutations of the pRpMatE>39 plasmid, respectively. The primers to introduce the point mutation C106A in the pRpMatE>39 plasmid are 5'-ccgaagatcgtggtggccgatccgtccaagcg-3' and 5'- cgcttggacggatcggccaccacgatcttcgg-3'. The primers to introduce R286C and Q457C, i.e. the mutations in the most preferred RpMatB biosensor, are respectively 5'- gctcgccgatacgcattgcgaatggtcg-3' and 5'-acgatcgacgaagcgtgcgtgctgcacggcctc-3'.
Protein expression and purification
RpMatB variants were synthesized in E. coli OverExpress C4i(DE3) cells (Lucigen). The cell pellet of a 5 ml overnight culture, grown at 37 °C and supplemented with 100 μg ml 1 ampicillin, was resuspended in 0.5 1 of lysogeny broth medium, supplemented with ampicillin, in 5 1 shaker flasks and cultured via vigorous shaking at 30 °C to an optical density at 600 nm of 0.6 - 0.8 cm 1. Then, 0.5 mM isopropyl- -D- thiogalactoside was added to start induction at 30 °C for 16 h. After induction, the cultures were cooled down to 4 °C and centrifuged at 3500 rpm for 30 min at 4 °C (rotor JS 4.2, Beckman). The cell pellet was washed with 30 ml of ice-cold buffer (10 mM Tris HCl pH 7.5, 300 mM NaCl), centrifuged at 3500 rpm for 30 min at 4 °C (rotor JS 4.2, Beckman), the supernatant discarded and the pellet stored at -80 °C until use. About 3 g wet weight of E. coli cells were harvested from 0.5 1 culture in a typical preparation.
The cell pellet was resuspended in 35 ml 30 mM Tris HCl, 300 mM NaCl, 10 mM imidazole, 3 mM tris(2-carboxyethyl)phosphine, 2 mM phenylmethanesulfonyl fluoride, pH 8.0 and sonicated on ice using an ultra-sonicator (VC505, Sonics) at 200 W for 5 times 30 s with a 5 s on / 5 s off pulser. The soluble fraction was collected by centrifugation at 35000 rpm for 45 min at 4 °C (rotor 45 Ti, Beckman). The His6-tagged protein was purified at 4 °C on an immobilized metal ion affinity chromatography (1 ml HisTrap HP column, GE Healthcare) using an Akta system (GE Healthcare). The resin was equilibrated with Buffer A (30 mM Tris HCl, 300 mM NaCl, 10 mM imidazole, 1 mM tris(2-carboxyethyl)phosphine, pH 8.0). The sample was filtered (0.45 μιη Minisart NML filter, Sartorius) and loaded onto the column at 0.5 ml min 1. The column was washed with 20 ml Buffer A and additionally with 20 ml of 95 % Buffer A and 5 % Buffer B (30 mM Tris HCl, 300 mM NaCl, 250 mM imidazole, 1 mM tris(2- carboxyethyl)phosphine, pH 8.0) at a flow rate of 1 ml min 1. The protein was eluted with 20 ml of Buffer B at a flow rate of 1 ml min 1. Protein fractions were pooled (~2-4 ml) and further purified via size exclusion chromatography at 4 °C using the HiLoad 16/60 Superdex 200 prep grade column (GE Healthcare) equilibrated with 30 mM Tris HCl, 100 mM NaCl, 0.5 mM ethylenediaminetetraacetic acid, 5 mM dithiothreitol, 1 mM NaN3. The flow rate was 1 ml min 1 and the loading volume ranged from 2 to 4 ml. Fractions containing the protein were pooled and concentrated (VivaSpin 20 MWCO 10 kDa cut off, GE Healthcare) to ~io mg ml 1.
The protein concentration was determined from the absorbance at 280 nm using the extinction coefficient at 280 nm of 46300 M 1 cm 1 calculated from the sequence via Expasy Protparam (Wilkins., Gasteiger et al. 1999). The protein was drop-frozen in liquid nitrogen and stored at -80 °C. Typically, 45 mg of protein was obtained from 3 g wet weight of cells.
Labeling of RpMatB with fluorescent dyes
We will first describe the detailed procedure for labeling and purification of the most preferred sensor variant RpMatB (His6-RpMatB C106A/R286C/Q457C/K488A). Deviations from this procedure during labeling and purification of RpMatB variants at previous stages of the sensor development are mentioned afterwards.
Dithiothreitol was removed from ~40 mg of protein using a PD10 desalting column (GE Healthcare) pre- equilibrated with Buffer L (30 mM Tris HCl pH 7.5, 100 mM NaCl) at 20 °C. 50 μΜ protein was incubated at 20 °C with 225 μΜ 5- iodoacetamidotetramethylrhodamine (5-IATR, AnaSpec, CA) in Buffer L using an end- over-end mixer for 90 min. Afterwards, 2 mM sodium-2-mercaptoethanesulfonate was added and incubation continued for 15 min. After centrifugation at 3500 rpm for 15 min at 4 °C (Heraeus Biofuge), the supernatant was filtered through a 0.2 μπι syringe filter (Acrodisc Syringe Filter with HT Tuffryn Membrane, Pall Life Sciences) and loaded onto a PD10 desalting column, equilibrated with Buffer Qi (30 mM Tris HCl pH 8.0, 25 mM NaCl) at -20 °C to remove free label.
The most preferred RpMatB biosensor was further purified via ion exchange chromatography at 4 °C using a 1 ml HiTrap Q HP column (GE Healthcare), equilibrated in Buffer Qi. The flow rate was 1 ml min 1 during the whole purification. After sample loading, the column was washed with 90 ml Buffer Qi. The protein was eluted using a gradient from 100 % Buffer Qi to 50 % Buffer Qi and 50 % Buffer Q2 (30 mM Tris HC1 pH 8.0, l M NaCl) over 25 ml followed by a gradient from 50 % Buffer Qi and 50 % Buffer Q2 to 100 % Buffer Q2 over 10 ml. Fractions containing the protein were pooled and concentrated to ~5 mg ml 1 using a concentrator (Amicon Ultra-4 10 kDa cut off, Millipore).
While screening for a fluorescence change upon ATP binding, labeling was performed on a scale of ~2 mg of protein. RpMatB variants (100 μΜ) were incubated at 20 °C with 2-fold (7-diethylamino-3-((((2-maleimidyl)ethyl)amino)carbonyl)coumarin (MDCC) or 7-diethylamino-3-((((2-iodoacetomido)ethyl)amino)carbonyl)coumarin (IDCC)) or 4- fold (5-IATR or 6-IATR (synthesized in-house (C rno and Craik : Q )) ) excess of fluorophore over RpMatB for 90 (tetramethylrhodamine), 35 (MDCC) or 120 (IDCC) min. During screening of all the labeled RpMatB variants, proteins were not further purified via ion exchange chromatography but tested after removal of free label using a PD10 desalting column.
The labeled protein concentrations were determined using the following extinction coefficients: RpMatB: 828onm (46300 M"1 cm 1), tetramethylrhodamine: 828onm (31000 M" 1 cm"1) and 8528nm (52000 M"1 cm"1) (Conic and Cr;;ik 0 4). MDCC: 828onm (7470 M"1 cm- and 843onm (46800 M"1 cm"1) and IDCC: 828onm (7470 M"1 cm"1) and 843onm (44800 M"1 cm 1). The protein was drop-frozen in liquid nitrogen and stored at -80 °C. Labeling yields were up to 35 %.
Using mass spectrometry, unlabeled RpMatB (His6-RpMatB C106A/R286C/Q457C/K488A) had a mass of 57335.9 Da. The theoretical molecular weight is 57324.2 Da, assuming loss of the N-terminal methionine. Presumably, other post-translational modifications, such as partial oxidation of surface-accessible methionines, occur (Gtian..Yate„s..e . :..20.Q ). The most preferred RpMatB biosensor, Rho-MatB ((5-ATR)2-His6-RpMatB C106A/R286C/Q457C/K488A)), had a mass of 58216.4 Da, conforming to the theoretical molecular weight of the RpMatB protein labeled with two 5-IATRs (57335.9 + 2 x 441.5).
The solution molecular weight was analyzed using size exclusion chromatography coupled to multi-angle laser light scattering (SEC-MALLS). Protein (1 mg ml 1) was applied in a volume of 100 μΐ to a Superdex 200 10/300 GL column (GE Healthcare) connected to a Jasco PU-1580 HPLC at a flow rate of 0.5 ml min 1. The HPLC system was connected to a Dawn Heleos II light scattering instrument (Wyatt Technology) and Optilab T-rex differential refractometer (Wyatt Technology). The solution molecular weight was determined from the combined data from both detectors using the ASTRA software version 6.1.1.17 (Wyatt Technology) with the refractive index increment set to 0.1860 ml g 1. RpMatB activity assay
RpMatB specific activity was quantified using a nicotinamide adenine dinucleotide (NADH) assay CCrosby^ I ank et aL.2012). Reaction mixtures (200 μΐ) contained 50 mM Hepes buffer pH 7.5, 25 mM NaCl, 10 mM MgCl2, 25 mM KC1, 0.3 mg ml 1 bovine serum albumin, 0.5 mM ATP, 0.5 mM coenzymeA, 3 mM phosphoenolpyruvate, 0.2 mM NADH, 0.01 U μΐ 1 pyruvate kinase (rabbit muscle (Sigma)), 0.05 U μΐ 1 adenylate kinase (chicken muscle (Sigma)), 0.015 U μΐ 1 lactate dehydrogenase (rabbit muscle (Sigma)) and 2 mM malonate. All reactions were started by the addition of RpMatB (0.03 μΜ), and the change in A34onm was monitored for several min at 25 °C on a Jasco V-550 UV-Vis Spectrophotometer (Jasco).
Fluorescence measurements
Fluorescent measurements were obtained on a Cary Eclipse Spectrofluorometer (Agilent Technologies), using a 3-mm path-length quartz cuvette (Hellma), unless otherwise mentioned. Excitation and emission slits were set at 5 nm. Protein and nucleotide concentrations, buffer conditions and excitation and emission wavelengths used are given in the figure legends.
For titrations to measure nucleotide binding, the fluorescence was excited at 553 nm and emission was measured at 571 nm. Data were analyzed with a quadratic binding curve using Grafit software (Leatherbarrow 200Q):
Figure imgf000055_0001
where P and L are the total concentrations of protein and ligand, respectively, Kd is the dissociation constant, and Fm in and Fmax are the fluorescence intensities of the free and ligand- bound protein, respectively.
Stopped-flow measurements
Stopped flow experiments were carried out using a HiTech SF-61DX2 apparatus (TgK Scientific, Bradford-on-Avon, UK) with a xenon-mercury lamp and operated by Kinetic Studio software (TgK Scientific). The excitation wavelength was 548 nm and there was an OG570 cut-off filter on the emission. The concentrations mentioned in the text and figures are those in the mixing chamber. Data were fitted to theoretical equations using the Kinetic Studio software. KinTek Global Kinetic Explorer software (version 4.0) (Johnson,. Simpson.ei.al...200.Q, Johnson. Simpson et al. 2009) was used to obtain global fits and to generate simulation data for possible kinetic schemes. Steady-state analysis of ATP production by pyruvate kinase
Absorbance: Steady-state activity measurements of pyruvate kinase (rabbit muscle from Sigma) were obtained on a CLARIOstar microplate reader (BMG Labtech) using a 96-well polystyrene microplate (black, clear flat bottom, Corning). Reaction mixtures (200 μΐ) contained 50 mM Tris HCl pH 7.5, 100 mM NaCl, 10 mM MgCl2, 100 mM KCl, 0.3 mg ml 1 bovine serum albumin, 250 μΜ ADP, 0.2 mM NADH, 5 U ml 1 lactate dehydrogenase and various phosphoenolpyruvate concentrations. All reactions were started by the addition of pyruvate kinase (0.1 U ml 1), and the change in A34onm was monitored for at 20 °C. The initial rates were obtained by linear regression, using an 834onm for NADH of 6220 M"1 cm"1.
Fluorescence: Steady-state measurements of pyruvate kinase activity were obtained on a CLARIOstar microplate reader (BMG Labtech) using a 96-well polystyrene microplate (black, F-bottom, Greiner). Reaction mixtures (200 μΐ) contained 50 mM Tris HCl pH 7.5, 100 mM NaCl, 10 mM MgCl2, 100 mM KCl, 0.3 mg ml 1 bovine serum albumin, 250 μΜ ADP, 2.5 μΜ Rho-MatB and various phosphoenolpyruvate concentrations. The emission at 580 nm (10 nm bandwidth) after excitation at 545 nm (10 nm bandwidth) was recorded. A calibration curve was determined using various concentrations of ATP added to the solution above in the presence of 2.5 mM phosphoenolpyruvate. Reactions were started by the addition of pyruvate kinase (0.025 U ml 1), and the change in fluorescence signal was monitored at 20 °C. Linear regression analysis was used to determine the initial velocity.
Stopped- flow : Steady-state measurements of pyruvate kinase activity were obtained using a stopped-flow apparatus, as above. The excitation wavelength was 548 nm and there was an OG570 cut-off filter on the emission. Reaction mixtures contained 50 mM Tris HCl pH 7.5, 100 mM NaCl, 10 mM MgCl2, 100 mM KCl, 0.3 mg ml 1 bovine serum albumin, 250 μΜ ADP, 100 μΜ phosphoenolpyruvate and 2.5 μΜ Rho-MatB. A calibration curve was determined using various concentrations of ATP added to the solution above. Reactions were started by the addition of pyruvate kinase (up to 6.0 U ml 1) and the change in fluorescence signal was monitored for several seconds at 25 °C. Linear regression analysis was used to determine the initial velocity. Exam ple 1: Design of the biosenso r, base d on RpMatB
Finding a suitable candidate protein recognition element for an ATP biosensor requires comparing ATP- or ATP-analogue- bound protein structures with their corresponding ligand-free protein structures. If that comparison revealed a significant conformational change upon ligand binding, the protein was seen as a potential candidate for biosensor development. Such conformational changes can be harnessed to transduce ligand binding to a fluorescence change to a fluorophore reporter, local to that region of the protein so that it responds to the change in structural environment. Functional parameters were considered next, for example affinity and selectivity for ATP, or known mutants that block enzymatic activity, such as ATPase activity. Following this analysis, RpMatB was chosen as the most suitable candidate for further ATP-biosensor development.
The crystal structures for the apo and MgATP-bound states of RpMatB suggested that there is a conformational change upon MgATP-binding (Crosby., Rank et al. 2012). In particular, the C-terminal domain rotates ~20° towards the N-terminal domain closing the active site cleft (Figure lA and Figure lB).
Cysteine mutations were introduced as sites for labeling onto a background of the C106A and K488A mutations in the wild-type protein. The K488A RpMatB variant does not catalyze the adenylation half-reaction, that is it cannot convert ATP and malonate to malonyl-AMP and pyrophosphate (Cro.sj3 5..R k. ...aI^.2Qi2). C106 in the wild-type protein (Figure lA) is situated in the N-terminal domain, distant from the active site but slightly solvent accessible. Having shown that there is a low, but significant, degree of background labeling at this position (6% with MDCC), C106 was mutated to alanine.
Several variants were designed and prepared with either one or two cysteine residues introduced in order to label C106A/K488A RpMatB with one diethylaminocoumarin or two tetramethylrhodamines, respectively.
After examination of the ligand-bound and ligand-free RpMatB structures, sites for diethylaminocoumarin labeling were chosen, situated around the ligand-binding pocket on the C-terminal lobe, so that the fluorophore might experience an environmental change when MgATP binds. These labeled variants were tested for the fluorescence change upon ATP -binding in the presence of Mg2+ (Supplemental Table 1).
Sites for tetramethylrhodamine labeling were chosen so that stacking of the two fluorophores might be possible in the apo conformation and that dissociation of these stacked tetramethylrhodamines could occur on the conformational change with MgATP binding. In particular, positions were chosen with a suitable distance (-1.5 nm) and orientation between them in the apo conformation. In addition, the distance and orientation between the chosen positions changed when MgATP binds. Results from seven different combinations are in Supplemental Table 1.
The labeled variant with the largest fluorescent increase upon MgATP-binding was (5- ATR)2-His6-RpMatB C106A/R286C/Q457C/K488A, hereinafter referred to as Rho- MatB. Both positions are well defined in the apo and MgATP-bound structure as shown in Figure lA. R286 is located in the N-terminal domain and Q457 in the C-terminal domain.
Exam ple 2 : Fluorescence and absorbance properties of Rho-MatB with ATP Figure 2A shows fluorescence excitation and emission spectra of Rho-MatB upon addition of increasing amounts of ATP. The fluorescence spectral shape did not change, but the fluorescence intensity increased 3.7- fold.
Absorbance spectra of Rho-MatB were also measured for a range of ATP concentrations (Figure 2B). In the absence of nucleotide, the maximum absorbance was at 518 nm with a smaller peak at 553 nm. This is characteristic of tetramethylrhodamine stacking, corresponding qualitatively to the absorbance spectra for other stacked rhodamines (Seiwyn and Stemiel.Ji 1972, Chambers, ajiwara et al. 1974). Binding ATP causes an absorbance decrease at 518 nm and increase at 553 nm, indicating that nucleotide binding reduces the stacking, as monomeric rhodamine had a larger peak at ~550 nm. The isosbestic point was at 532 nm.
The affinity for ATP was determined by measuring the fluorescence at different concentrations of ATP in a solution of Rho-MatB and ¾,A P was 6.4 μΜ (Figure 3A).
Exam ple 3 : Affinity of Rho-MatB for other nucleotides and potential ligands
The fluorescence of Rho-MatB responded to addition of nucleotides other than ATP. There was an increase in fluorescence intensity upon addition of ADP, deoxyadenosine triphosphate (dATP), adenosine 5'-(Y-thio)triphosphate (ATPyS) and adenosine 5'-(β,γ- imido)triphosphate (AMP-PNP). In contrast, AMP, GDP and GTP did not have a significant effect. Also, RpMatB substrates other than ATP (i.e. malonate and coenzymeA) had no influence on the fluorescence and their presence did not inhibit the MgATP-induced fluorescence increase (Supplemental Figure 2A).
The affinity for the responsive nucleotides, namely ADP, dATP, ATPyS and AMP-PNP was determined by measuring the fluorescence at different concentrations of the nucleotide in a solution of Rho-MatB (Figure 3A and Table 1). The dissociation constants for ADP, dATP, ATPyS and AMP-PNP were 428 μΜ, 440 μΜ, ι6.2 μΜ and 253 μΜ, respectively. So the binding of ADP, dATP and AMP-PNP to RpMatB was much weaker than ATP. The affinity of ATPyS to Rho-MatB was similar to ATP. The maximum fluorescence increase upon binding these nucleotides was smaller than with ATP.
In most biological reactions ATP is likely to be produced from ADP. Consequently, ADP will be present in the assay solutions, so the influence of ADP on the fluorescence signal change and the affinity of ATP was assessed. Titration curves for ATP were obtained in the presence of a fixed concentration of ADP. The apparent ¾ for ATP was similar in the absence and presence of 100 μΜ ADP (Figure 3B and Table 1).
In addition, the influence on ATP affinity of ATP analogues was assessed. Titration curves for ATP were obtained in the presence of a fixed concentration of dATP, ΑΤΡγβ and AMP-PNP. Only ATPyS had a remarkable influence on the apparent Ka for ATP by increasing it ~5-fold (Figure 3B and Table 1).
It simplifies application of a fluorescence biosensor, if it can be used over a range for which the response to concentration is approximately linear. Figure 3C shows there is a good linear fit up to 6 μΜ ATP. The fluorescence response to ATP was also measured in the presence of different ADP concentrations but at constant total nucleotide concentration to mimic ADP conversion to ATP. Although the slope decreased with increasing ADP concentration, the fluorescence response to ATP remained linear in the presence of ADP over the same range (Figure 3C).
Exam ple 4 : The influence of so lutio n conditions on the fluorescent pro perties of Rho -MatB
The fluorescence response to ATP was measured in different buffers, over a pH range from 6.0 to 9.0 and different ionic strengths with salt from 50 to 200 mM (Supplemental Table 2). Although the size of the fluorescence response changed with solution conditions, there were rather small effects on the ¾ for ATP.
Mg2+ is required for the fluorescence change, suggesting that MgATP is bound (Supplemental Figure 2B), as might be expected as Mg2+ is a cofactor for the enzyme. Exam ple 5 : Binding kinetics
The kinetics of ATP binding and dissociation were measured by stopped-flow fluorescence in order to assess the range of rates over which Rho-MatB is suitable for real-time measurements.
Binding kinetics were first measured under pseudo-first-order conditions by rapidly mixing different concentrations of ATP, in large excess, with Rho-MatB (Figure 4A & 4B). The time course of the subsequent fluorescence response was biphasic, fitting well to a double exponential. The observed rate constant for the fast phase increased linearly up to 150 μΜ ATP (Figure 4C), giving a slope of 1.83 μΜ 1 s 1 and the intercept was 8.2 s 1. Assuming this phase represents binding, the slope is the association rate constant, the intercept is the dissociation rate constant. The dissociation constant, calculated from these values (4.5 μΜ) agrees well with the value from equilibrium binding data (Figure 3A and Table 1). The observed rate constants for the slow phase did not vary significantly over the ATP concentration range with an average value of 0.88 s 1. The biochemical basis of these rate constants will be discussed later.
In order to explain the biphasic kinetic data for ATP binding in excess ATP, the data were fitted to two possible two-step binding mechanism as shown in Figure 5. In the first model a pre-existing conformational equilibrium in the apoprotein was followed by association of MgATP to one of the conformations (conformational selection, Figure 5A). The second model involves a structural transition that follows MgATP-binding (induced fit, Figure 5B). Both mechanisms were possible.
To dissect between the two schemes, simulations of nucleotide-binding experiments using excess Rho-MatB were performed using KinTek Global Kinetic Explorer. By conducting simulations at varying initial concentrations of excess Rho-MatB for both mechanisms, single phase binding kinetics in the case of conformational selection and biphasic binding kinetics in the case of induced fit was observed (Supplemental Figure 3A and Supplemental Figure 3B).
ATP binding was measured with Rho-MatB present in large excess over the nucleotide. Rapid mixing produced a monophasic, exponential increase over the whole range of Rho-MatB concentrations used (Figure 4D). The observed rate constant increased linearly with [Rho-MatB], giving an association rate constant of 0.72 μΜ 1 s 1 and the intercept was 8.0 s 1, similar to the values obtained in the excess of ATP (Figure 4E). Hence, only the conformational selection mechanism fits our data.
Dissociation kinetics were measured directly starting from Rho-MatB.ATP complex and trapping dissociated ATP with a large excess of unlabeled protein (Figure 4F). The fluorescence decreased with time and the curves required a double exponential with rates 6.9 s 1 and 1.5 s 1. These do not vary when the concentration of the unlabeled protein varies, indicating that the process is ATP-dependent.
Exam ple 6 : Enzym atic activity
In order to confirm that the most preferred RpMatB construct lacked enzymatic activity, as expected as it contains K488A, ATPase activity was measured using a coupled-enzyme assay with NADH consumption (Crosby. Ran k ;.·[ . 201:2 ). Rho-MatB showed no residual activity under the conditions tested (Supplemental Figure 4). In comparison, the similar Rho-MatB variant but without the K488 mutation ((5-ATR)2- His6-RpMatB C106A/R286C/Q457C) had a specific activity of 37 ± 4 μηιοΐ AMP mg 1 min 1.
Exam ple 7: Test assay: Steady-state production of ATP by pyruvate kinase The ATP biosensor was tested in a steady-state assay in which ATP was produced from ADP and phosphoenolpyruvate in a reaction catalyzed by pyruvate kinase (Figure 6A and Figure 6B). This was chosen as there is a coupled-enzyme assay for this enzyme, that is well established and described below, which could be used to validate the biosensor results. The rate of ATP formation was measured at different concentrations of phosphoenolpyruvate using Rho-MatB. The resulting fluorescence traces were analyzed, using a calibration curve of fluorescence between o and 5 μΜ ATP, obtained under similar experimental conditions. Rates of ATP formation were plotted as a function of phosphoenolpyruvate concentration and fitted to Michaelis-Menten kinetics (Figure 6C), giving a Km of 103 μΜ and Vmax of 0.76 μΜ s 1 U 1 ml 1.
As a comparison, pyruvate formation was measured using a coupled-enzyme assay under the same conditions to compare the parameters directly. This coupled the pyruvate kinase reaction to that of lactate dehydrogenase, in which NADH reduces the pyruvate as it is converted to NAD+. This assay gave a Km of 251 μΜ and a Vmax of 0.66 μΜ s 1 U 1 ml 1 (Supplemental Figure 5). The Vmax was similar to that obtained using Rho-MatB; reasons why the Km is different for the two assays probably relates to the different extents of reaction required to perform the two assays.
In order to test the ability of Rho-MatB to assay more rapid ATP production, measurements were done with higher concentrations of pyruvate kinase. Because the time scale was below that of manual mixing, the reactions were done in a stopped flow apparatus (Figure 6D). The observed rate was proportional to the amount of pyruvate kinase added (Figure 6E).
Exam ple 8 : Annotated RpMatB se quence Figure 17 shows an exemplary annotated RpMatB sequence.
Numbering scheme and position of mutations:
The N-terminal His6-tag is highlighted in vzdf italics. The mutations are highlighted in blue/ underlined and labeled. The residues in close proximity (sequence and space) of the labeled positions are highlighted in gt*e *x/ bold. They are summarised in Table 2. Suitably truncated forms are those which lack a small number of amino acid residues from the N- or C- terminus of the polypeptide relative to wild type MatB.
Exam ple 9
Design of the biosensor After deciding that RpMatB was a suitable recognition element for ATP, we introduced either one or two cysteine residues in order to label RpMatB with diethylaminocoumarin or tetramethylrhodamine. We tested all variants for their fluorescence change upon MgATP addition and found different fluorescence responses to MgATP binding, ranging from a decrease to an increase in fluorescence intensity (Supplemental Table 1).
Hence, not all fourteen rationally designed RpMatB mutants had identical properties, probably due to a combination of several factors. However, those having the desired and most advantageous properties could be identified as described.
Without wishing to be bound by theory, reasons for the range of observations may include:
Firstly, introducing a fluorophore might have an effect on protein flexibility and therefore on the documented conformational change upon ligand binding (either because of interaction with amino acids or because of interactions between fluorophores).
Secondly, whereas comparisons between apo and ATP (analogue) -bound structures indicate that most of the ANL superfamily members undergo a conformational change after ATP (analogue) binding, we observed that the interdomain geometry in apo structures of ANL superfamily members is less conserved than in ATP (analogue) - bound structures (Supplemental Table 3). In addition, several observations provide evidence that the C-terminal domain is also more flexible than the N-terminal domain. This is based on the absence of the C-terminal domain in a lot of apo structures within the ANL superfamily and the higher average B-factor for the C-terminal domain compared to the N-terminal domain in crystal structures of proteins from the ANL superfamily (Supplemental Table 3). Both findings, namely the structurally less conserved apo conformation and a flexible C-terminal domain, would interfere with our rational design. Characterization of Rho-MatB
Rho-MatB can be used as a biosensor for ATP under various pH and salt conditions (Supplemental Table 2) in the presence of Mg2+ (Supplemental Figure 2B). The maximum fluorescence increase upon ATP-binding was 3.7-fold (Figure 2A). The change in the absorbance spectrum provides support that this increase is due to the unstacking of the two tetramethylrhodamines upon ATP-binding (Figure 2B).
Rho-MatB binds ATP with a dissociation constant of 6.4 μΜ (Figure 3A and Table 1), higher than RpMatB K488A (0.31 μΜ) CCm^ :a... nk..et..d-...2Qi2). This difference mmiigghhtt bbee ccaauusseedd bbyy tthhee ppooiinntt mmuuttaattiioonnss iinnttrroodduucceedd oorr tthhee tteettrraammeetthhyyllrrhhooddaammiinnee f flluuoorroopphhoorreess aattttaacchheedd ttoo RRppMMaattBB..
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TThhee pprreeffeerrrreedd RRhhoo--MMaattBB bbiioosseennssoorr eexxhhiibbiitteedd aa llooww ddeetteeccttiioonn lliimmiitt ((--00..2255 μμΜΜ)).. BBiinnddiinngg kkiinneettiiccss
1155 TThhee AATTPP bbiioosseennssoorr rreessppoonnddss rreellaattiivveellyy ffaasstt ttoo AATTPP.. BBiipphhaassiicc bbiinnddiinngg kkiinneettiiccss bbeettwweeeenn RRhhoo--MMaattBB aanndd AATTPP wweerree oobbsseerrvveedd iinn eexxcceessss AATTPP ((FFiigguurree 44BB))..
TThhee ffaasstt pphhaassee iiss AATTPP--ddeeppeennddeenntt -- iinn aa lliinneeaarr wwaayy ((FFiigguurree 44CC));; tthhee ssllooww pphhaassee nnoott.. LLiinneeaarr ccoonncceennttrraattiioonn ddeeppeennddeennccee ooff tthhee oobbsseerrvveedd bbiinnddiinngg rraatteess iinnddiiccaatteess ssiimmppllee ffiirrsstt-- oorrddeerr bbiinnddiinngg.. HHoowweevveerr,, wwee ddoo hhaavvee ttoo ppooiinntt oouutt tthhaatt tthheerree iiss aa ddeevviiaattiioonn ffrroomm lliinneeaarriittyy 2200 ffoorr kk00bbss,,ffaasstt aatt hhiigghheerr AATTPP ccoonncceennttrraattiioonnss,, ssuuggggeessttiinngg ttwwoo--sstteepp bbiinnddiinngg.. HHoowweevveerr,, nnoo vvaalliidd rraattee ccoonnssttaannttss ccaann bbee oobbttaaiinneedd ffoorr AATTPP ccoonncceennttrraattiioonnss ooff mmoorree tthhaann 220000 μμΜΜ aatt 2255 °°CC dduuee ttoo tthhee ddeeaadd ttiimmee ooff tthhee iinnssttrruummeenntt.. TThheerreeffoorree,, nnoo aaccccuurraattee kkiinneettiicc ppaarraammeetteerrss ffoorr aa ttwwoo--sstteepp bbiinnddiinngg mmeecchhaanniissmm aarree aavvaaiillaabbllee..
AAtt tthhiiss ppooiinntt,, wwee pprreessuummeedd tthhee ssllooww pphhaassee ccoouulldd bbee ccaauusseedd eeiitthheerr bbyy aa rraattee--lliimmiittiinngg 2255 ccoonnffoorrmmaattiioonnaall cchhaannggee pprriioorr ttoo AATTPP--bbiinnddiinngg ((ccoonnffoorrmmaattiioonnaall sseelleeccttiioonn,, FFiigguurree 55AA)) oorr bbyy aa ssttrruuccttuurraall ttrraannssiittiioonn tthhaatt ffoolllloowwss MMggAATTPP--bbiinnddiinngg ((iinndduucceedd ffiitt,, FFiigguurree 55BB)) ((VVooggLL
Figure imgf000063_0001
To dissect between the two schemes, we carried out nucleotide-binding experiments using excess Rho-MatB. Since simulations conducted at varying initial concentrations
30 of excess Rho-MatB predicted single phase binding kinetics in the case of conformational selection and biphasic binding kinetics in the case of induced fit (Supplemental Figure 3A and Supplemental Figure 3B). We observed single phase binding kinetics (Figure 4D) and the observed rate constant is Rho-MatB-dependent (Figure 4E). This corresponds to the conformational selection mechanism (Figure 5A).
35 Therefore, we believe that Rho-MatB exists in two different conformations prior to ATP-binding. ATP binds to only one conformation. In excess Rho-MatB, there is "enough" Rho-MatB in the "correct" conformation to bind ATP and we observe only AATTPP--bbiinnddiinngg,, hheennccee ssiinnggllee pphhaassee bbiinnddiinngg kkiinneettiiccss.. IInn eexxcceessss AATTPP,, tthheerree iiss nnoott ""eennoouugghh"" RRhhoo--MMaattBB iinn tthhee ""ccoorrrreecctt"" ccoonnffoorrmmaattiioonn ttoo bbiinndd aallll AATTPP iimmmmeeddiiaatteellyy aanndd wwee oobbsseerrvvee AATTPP--bbiinnddiinngg aanndd tthhee RRhhoo--MMaattBB ccoonnffoorrmmaattiioonnaall cchhaannggee..
OOff nnoottee,, eevveenn tthhee ccoonnffoorrmmaattiioonnaall sseelleeccttiioonn sscchheemmee ddooeess nnoott eexxaaccttllyy ffiitt aallll eexxppeeririmmeennttaall 55 rreessuullttss.. AAddddiittiioonnaall sstteeppss ttoo tthhee sscchheemmee mmiigghhtt bbee rreeqquuiirreedd,, ffoorr eexxaammppllee aa mmuullttii--sstteepp bbiinnddiinngg iinn wwhhiicchh bbootthh ccoonnffoorrmmaattiioonnaall sseelleeccttiioonn aanndd iinndduucceedd ffiitt aarree pprreesseenntt.. HHoowweevveerr,, ffiinnddiinngg aa ddeettaaiilleedd mmeecchhaanniissmm ffoorr RRhhoo--MMaattBB iiss nnoott tthhee ssccooppee ooff tthhiiss ddooccuummeenntt.. WWee hhaavvee ddeessccrriibbeedd aa mmiinniimmaall mmooddeell ttoo ffiitt aallll ooff tthhee ddaattaa..
WWee hhaavvee ttoo aadddd tthhaatt aa rraattee--lliimmiittiinngg ccoonnffoorrmmaattiioonnaall cchhaannggee pprriioorr ttoo AATTPP--bbiinnddiinngg 1100 ((ccoonnffoorrmmaattiioonnaall sseelleeccttiioonn)) iiss ccoonnssiisstteenntt wwiitthh tthhee SSEECC--MMAALLLLSS ddaattaa tthhaatt cclleeaarrllyy sshhooww ttwwoo ddiiffffeerreenntt ssppeecciieess iinn tthhee aabbsseennccee ooff AATTPP ((SSuupppplleemmeennttaall FFiigguurree 66))..
TThhee sseennssoorr wwoorrkkss wweellll aanndd aa ppoossiittiivvee ddeemmoonnssttrraattiioonn ooff iittss aaccttiioonn iiss pprroovviiddeedd hheerreeiinn..
UUssee ooff tthhee bbiioosseennssoorr
1155 TThhee AATTPP bbiioosseennssoorr wwaass tteesstteedd iinn aa sstteeaaddyy--ssttaattee aassssaayy iinn wwhhiicchh AATTPP wwaass pprroodduucceedd ffrroomm AADDPP aanndd pphhoosspphhooeennoollppyyrruuvvaattee iinn aa rreeaaccttiioonn ccaattaallyyzzeedd bbyy ppyyrruuvvaattee kkiinnaassee ((FFiigguurree 66)).. IInn aaddddiittiioonn,, ppyyrruuvvaattee ffoorrmmaattiioonn wwaass mmeeaassuurreedd uussiinngg aa ccoouupplleedd--eennzzyymmee aassssaayy wwiitthh llaaccttaattee ddeehhyyddrrooggeennaassee ttoo ccoommppaarree tthhee ppaarraammeetteerrss ddiirreeccttllyy ((SSuupppplleemmeennttaall FFiigguurree 55)).. WWhhiillee tthhee VVmmaaxx wwaass ssiimmiillaarr,, tthhee KKmm wwaass ddiiffffeerreenntt ffoorr tthhee ttwwoo aassssaayyss.. AAccccoorrddiinngg ttoo uuss,, tthhiiss mmiigghhtt
2200 rreellaattee ttoo tthhee eexxtteenntt ooff rreeaaccttiioonn rreeqquuiirreedd ttoo ppeerrffoorrmm tthhee ttwwoo aassssaayyss.. IInn oonnee,, yyoouu mmeeaassuurree AATTPP pprroodduuccttiioonn bbyy ppyyrruuvvaattee kkiinnaassee ddiirreeccttllyy,, iinn tthhee ootthheerr,, yyoouu mmeeaassuurree ppyyrruuvvaattee pprroodduuccttiioonn tthhrroouugghh llaaccttaattee ddeehhyyddrrooggeennaassee..
AAddvvaannttaaggeess ccoommppaarreedd ttoo ootthheerr aassssaayyss ttoo mmoonniittoorr AATTPP pprroodduuccttiioonn iinn vviittrroo
2255 TThhee bbiioosseennssoorr ffaacciilliittaatteess mmeeaassuurriinngg AATTPP iinn vviittrroo wwiitthh rreellaattiivveellyy hhiigghh ttiimmee rreessoolluuttiioonn..
TThhiiss mmaakkeess iitt ssuuiittaabbllee ffoorr aa rraannggee ooff rreeaall--ttiimmee,, kkiinneettiicc aassssaayyss,, wwhhiicchh ccaann bbee ddoonnee iinn cclloossee ttoo pphhyyssiioollooggiiccaall ccoonnddiittiioonnss,, ffoorr eexxaammppllee ppHH aanndd iioonniicc ssttrreennggtthh.. AA mmaajjoorr aaddvvaannttaaggee ooff rreeaaggeennttlleessss bbiioosseennssoorrss ooff tthhee iinnvveennttiioonn iiss tthhaatt oonnllyy aa ssiinnggllee ssppeecciieess iiss rreeqquuiirreedd ttoo mmoonniittoorr tthhee aannaallyyttee,, iinn tthhiiss ccaassee AATTPP,, tthheerreebbyy mmiinniimmiizziinngg tthhee lliikkeelliihhoooodd ooff 3300 iinntteerrffeerreennccee bbyy aaddddeedd rreeaaggeennttss..
IItt iiss aallssoo vveerryy ssppeecciiffiicc ffoorr AATTPP rreellaattiivvee ttoo AADDPP.. PPrreeffeerrrreedd sseennssoorr RRhhoo--MMaattBB hhaass 6677--ffoolldd hhiigghheerr aaffffiinniittyy ffoorr AATTPP tthhaann AADDPP.. TThhee hhiigghheesstt ssppeecciiffiicciittyy ssoo ffaarr ffoorr aa pprrootteeiinn--bbaasseedd bbiioosseennssoorr wwaass mmeeaassuurreedd ffoorr AATTeeaamm ((3300--ffoolldd hhiigghheerr aaffffiinniittyy ffoorr AATTPP tthhaann AADDPP
Figure imgf000064_0001
Finally, tetramethylrhodamine has high photostability. Its fluorescence is unlikely to interfere with (or be affected by) the system being studied because of excitation around 550 nm end emission around 570 nm. Thus, this ATP biosensor has many advantages relative to other ATP assays.
To sum up, the present work shows that Rho-MatB can be used as a sensitive probe to measure ATP. It shows a linear response in the micromolar range and is selective for ATP. It can be used to elucidate the mechanisms of ATP production.
Exam ple 10 : ATP biosensors using polypeptide sequence from members of the ANL superfamily other than RpMatB
This protein family contains acyl- and aryl-coenzymeA synthetases, the adenylation domains of nonribosomal peptide synthetases and firefly luciferases. Structural information (see Table B below) suggests a similar ligand-mediated conformational change for family members, even if the structures of ANL superfamily proteins lacking ligands are highly variable in the position of the C-terminus. This may be exploited to generate a family of ATP biosensors with possibly different properties (such as ATP sensitivity, fluorescence signal) using protein engineering techniques, based on straightforward structure and sequence principles. This approach has been successfully applied in the past to construct a biosensor family based on bacterial periplasmic binding proteins (de Lorimier, R. M., J. J. Smith, M. A. Dwyer, L. L. Looger, K. M. Sali, C. D. Paavola, S. S. Rizk, S. Sadigov, D. W. Conrad, L. Loew and H. W. Hellinga (2002). "Construction of a fluorescent biosensor family." Protein Sci 11(11): 2655- 2675).
This may be achieved as follows:
1. Identify a protein from the ANL superfamily either with a similar sequence or with a known structure with a similar conformation to that of RpMatB.
2. Identify sites within this protein, to which reporter fluorophores can be attached, based on homology to RpMatB, either sequence or structural homology
a. Sequence-based, using bioinformatics software:
i. Align the sequence of the candidate with the sequence of RpMatB.
Of note, the quality of the alignment will improve when performing a multiple sequence alignment instead of a pairwise alignment.
ii. Compare the aligned sequences. Identify the amino acids in the candidate that are close to the labeling sites in RpMatB.
b. Structure-based, using software for protein structure analysis and visualization:
i. Align the structure of the candidate with the structure of RpMatB. Of note, the quality of the superposition will improve when performing a multiple structure alignment instead of a pairwise protein structure alignment.
ii. Compare the superposed structures. Identify the amino acids in the candidate that are close to the labeling sites, identified in RpMatB. In this case, 6 proteins from the A L superfamily (2 acyl- and aryl -coenzyme A synthetase members, 2 adenylation domains of nonribosomal peptide synthetase members and 2 firefly luciferase members) were chosen because crystal structures have been solved for the closed conformation. The selection was based on a review of the structures (Gulick, A. M. (2009). "Conformational dynamics in the Acyl-CoA synthetases, adenylation domains of non-ribosomal peptide synthetases, and firefly luciferase." ACS Chem Biol 4(10): 811-827, http://1ab8.b.wi..bufYalo,edu/gulick/table.htrnl). To identify sites within "the candidate" that undergo local conformational change and to which reporter functions can be attached, a structure-based approach has been applied to this set of 6 proteins, as in the Figure. This gives amino acids in each case that are in similar locations to the Cysteine mutation sites of RpMatB (first line of the Table)
TABLE B: ANL superfamily members identified for structural similarity with the closed conformation of RpMatB and the proposed mutation sites, based on structural comparison as in Figure 19
Figure imgf000066_0001
References to publications can be found using the links at the Protein Data Bank.
Suitably the polypeptide component of the ATP sensor of the invention comprises sequence corresponding to one of the polypeptides in the above table.
Exam ple 11: Location of alternate sites for labelling ATP sensors
Figure 20 shows cartoon representations of RpMatB. The N-terminal domain (amino acids 1-399) in the apo conformation (PDB 4FUQ (Crosby, Rank et al. 2012)) is coloured grey. The C-terminal domain (amino acids 400-503) in the apo conformation is shown in pink. The C-terminal domain in the MgATP-bound conformation (PDB 4FUT (Crosby, Rank et al. 2012), after superimposing the N-terminal domain of 4FUT on the N-terminal domain of 4FUQ) is shown in green. ATP (in ball and stick conformation and coloured by CPK convention) and Mg2+ (depicted as an orange sphere) are shown in the active site. The C-terminal domain rotation upon MgATP- binding is clearly visible.
The positions of mutations are shown as spheres and labeled:
(A) C106A in the N-terminal domain (yellow), K488A in the C-terminal domain (red), R286C in the N-terminal domain and Q457C in the C-terminal domain (both blue).
(B) C106A in the N-terminal domain (yellow), K488A in the C-terminal domain (red), R286C in the N-terminal domain and G461C in the C-terminal domain (both blue). (C) C106A in the N-terminal domain (yellow), K488A in the C-terminal domain (red), K385C in the N-terminal domain and K470C in the C-terminal domain (both blue).
(D) C106A in the N-terminal domain (yellow), K488A in the C-terminal domain (red) and N492C in the N-terminal domain (blue).
(E) C106A in the N-terminal domain (yellow), K488A in the C-terminal domain (red) and E439C in the N-terminal domain (blue).
Exam pie 12 : Alignment and sequence similarity of ANL superfamily
A search for potentially related sequences of known structure can be performed by the profile.buildO command of MODELLER (manual page https://Milab.org/modeller/manud/node404.html). A database containing all PDB sequences, as of October 16, 2014, was searched using the RpMatB wild type sequence. All the aligned sequences (166 in number) were reviewed. For clarity, the sequences / structures / alignments used in Table B and Figure 19 were extracted and information on these is given below. These all show similar lid domain closing as RpMatB.
Sequence similarity scores were calculated using the EMBOSS NEEDLE
(http.://ww^^
Sequence Sequence
Protein PDB ID
identity similarity #
RpMatB, an acyl- and aryl-coenzymeA 4FUT,
NA NA
synthetases chain A
Homo sapiens ACSM2A, an acyl- and aryl- 3C5E,
30 44 coenzymeA synthetases chain A
Streptomyces sp CytCl, an adenylation domain 3VNQ,
27 43 of nonribosomal peptide synthetase chain A
Photinus pyralis luciferin 4-monooxygenase, a 4G36,
27 41 firefly luciferase chain A
Burkholderia xenovorans benzoate-coenzyme
2V7B,
A ligase, an acyl- and aryl-coenzymeA 28 41 chain A
synthetases (light pink)
Bacillus cereus DltA, an adenylation domain of 3FCC,
21 34 nonribosomal peptide synthetase chain A
Luciola cruciata luciferin 4-monooxygenase, a 2D1Q,
29 44 firefly luciferase chain A # BLOSUM62 matrix, gapopen 10.0, gapextend 0.5, endopen 10.0, endextend 0.5, pairwise alignment. Suitably the polypeptide component of the ATP sensor of the invention comprises sequence corresponding to one of the polypeptides in the above table.
Sequence alignment was determined, using structure/sequence features and the salign command of MODELLER (manual page hups./ ! il l i ab o; u inot!ci i c; ΠΊ ηίΐ^ ί n dc i ? h s ;n i ).
_aln . pos 10 20 30 40 50 60
4futA MNANLFARLFD- -LDDPH LAIET- -AAGD- - ISY 3c5eA QWGHQEVPA KFNFASDVLDHWADMEKAGKRPPSPALWWVNGKGKELMWNF 3vnqA TSDPIARNSDLVSLFREVAATAP ERTALSA- -EDDR- - ISY 4g36A AK IKKGPAPFY PLEDGTAGEQLHKAMKRYAL VPGTIAFTDAHIEVN- - ITY 2v7bA LFNFAAYLFRLNETRA GKTAYID- -DTGS- -TTY 3fccA MKLLEQIEKWAAETP DQTAFVW- -RDAK- - ITY 2dlqA E-NIWGPKPFY PIEEGSAGTQLRKYMERYAK L-GAIAFTNAVTGVD- -YSY consrvd
_aln . p 70 80 90 100 110 120 130 4futA AELVARAGRVANVLVA-RGLQVGDRVAAQTE- SVEALVLYLATVRAGGVYLPLNTAYTLHELDYFITD 3c5eA RELSENSQQAANVLSGACGLQRGDRVAWLPRVPEWWLVILGCIRAGLIFMPGTIQMKSTDILYRLQM 3vnqA GRLDAWSDAVARTLLA-EGVRPGDRVALRMSPGAEAIVAILAILKCGAAYVPVDLRNPVSRSDFILAD 4g36A AEYFEMSVRLAEAMKR-YGLNTNHRIWCSENSLQFFMPVLGALFIGVAVAPANDIYNERELLNSMNI 2v7bA GELEERARRFASALRT-LGVHPEERILLVMLDTVALPVAFLGALYAGWPWANTLLTPADYVYMLTH 3fccA KQLKEDSDALAHWISS-EYPDDRSPIMVYGHMQPEMIINFLGCVKAGHAYIPVDLSIPADRVQRIAEN 2dlqA AEYLEKS- -LGKALQN-YGLWDGRIALCSENCEEFFIPVIAGLFIGVGVAPTNEIYTLRELVHSLGI
_consrvd
_aln . pos 140 150 160 170 180 190 200 4futA AEP-IWCDPS RDGIAAIAAVGATVETLGPDGR GSLTDAAAGAS EAFATIDRGA 3c5eA SKAKAIVAGDEVIQEVDTVASECPSLRIKLLVS-EKSCDGWLNFKKLLNEAS TTHHCVETGS 3vnqA SGASALIG--EP HEGCA VTRWRT AAVAECKD--A E APGPG 4g36A SQPTWFVSKKGLQKILNVQKKLPIIQKII IMDSKTDYQGFQSMYTFVTSHLPPGFNEYDFVPESFDR 2v7bA SHARAVIASGALVQNVTQALESAG- - CQLIVSQP LAPLFEELIDAAA PAAKAAATGC 3fccA SGAKLLLSATAV TVTDL PVRIVSE DNLKDIFFTHK GNTPNPEHAVK 2dlqA SKPTIVFSSKKGLDKVITVQKTVTTIKTIVILDSKVDYRGYQCLDTFIKRNTPPGFQASSFKTVEVDR
_consrvd
_aln . pos 210 220 230 240 250 260 270 4futA D-DLAAILYTSGTT-GRSGAMLSHDNLASNSLTLVDY- - -WRFTPDDVLIHALPIYHTHGLFVASNVT 3c5eA Q-EASAIYFTSGTSGLPKMAEHSYSSLGLKAKMDAG- - -WTGLQASDIMWTISDTGWILNILCSLMEP 3vnqA AEDMAYVIYTSGTTGNPKGVPVRHANVLALLAGAPSV- - - FDFSGDDRWLLFHSLSFDFSV-WEIWGA 4g36A LPKGVALPHRTACVRFSHARDPIFGNQIIPDTAILSWPFHHGFGMFT-TLGY 2v7bA KPKGTVHTHANLYWTAELYAKPILGIA- -ENDWFSAAKLFFAYGLGNGLTFP 3fccA - PKGVQITYNCLVSFTKWAVED FNLQTGQVFLNQAPFSFDLSV-MDIYPS 2dlqA LPKGVQLTHENIVTRFSHARDPIYGNQVSPGTAVLTWPFHHGFGMFT-TLGY
_consrvd
_aln . pos 280 290 300 310 320 330 340 4futA LFARGSMIFLP FDPD- ILDLMA- -RATVLMGVPTFYTRLLQSPRLT-ETTGHMRLFISGSAPLL 3c5eA WALGACTFVHLLP-KFDPLVILKTLSSYPIKSMMGAPIVYRMLLQQ-DLSSYKFPHLQNCVTVGESLL 3vnqA FSTGAELWLPHWAARTPEQYLAVIIDRGVTVINQTPTAFLALTEAAVRGGRDVSGLRYVIFGGEKLT 4g36A LICGFRWLM- -Y-RFEEELFLRSLQDYKIQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLS 2v7bA LSVGATAILMA-E-RPTADAIFARLVEHRPTVFYGVPTLYANMLVSPNLPARADVAIRICTSAGEALP 3fccA LVTGGTLWAIDKDMIARPKDLFASLEQSDIQVWTSTPSFAEMCLMEASFSESMLPNMKTFLFCGEVLP 2dlqA LICGFRWML- -T-KFDEETFLKTLQDYKCTSVILVPTLFAILNKSELLNKYDLSNLVEIASGGAPLS
_consrvd
_aln . pos 350 360 370 380 390 400
4futA ADTHREWSATGH AVLERYGMTET-NMNTSNPYDGDRV PGAVGPALPGVSARVTDPE 3c5eA PETLENWRAQTGL DIRESYGQTET-GLTCMVSKTMKIK PGYMGTAASCYDVQ11D-D 3vnqA APMLRPWAKAFGLDRPRLVNGYGITETTVFTTFE EITEAYLA-QDASI IGRALPSFGTRWG-D 4g36A KEVGEAVAKRFHLP- -GIRQGYGLTETTSAILITPE-GDDK PGAVGKWPFFEAKWDLD 2v7bA REIGERFTAHFGC EILDGIGSTEMLHIFLSNRA-GAVE YGTTGRPVPGYEIELRD-E 3fccA NEVARKLIERFP- -KATIMNTYGPTEATVAVTGI HVTEEVLDQYKSLPVGYCKSDCRLLIMK-E 2dlqA KEVGEAVARRFNLP- -GVRQGYGLTETTSAI11TPE-GDDK PGASGKWPLFKAKVIDLD
_consrvd
_aln . p 410 420 430 440 450 460 470 4futA TG-ELPRGDIGMIEV GPNVF-GYWRMPE- -TSEFRDD- - -GFFITGDLG- IDERGYVHILGR 3c5eA KGNVLPPGTEGDIGIRVKPIRPIGIFSGYVDNPDKTAANIRGD- - - -FWLLGDRGIKDEDGYFQFMGR 3vnqA DGRDVAPGETGELWLS GAQLAEGYLRRPELTAEKFPEW- -RYYRTGDLVSELPDGRFAYEGR 4g36A TGKTLGVNQRGELCVR GPMIMSGYVNNPEATNALIDKD- - -GWLHSGDIAYWDEDEHFFIVDR AGHAVPDGEVGDLYIK GPSAAVMYWNNREKSRATFL-G EWIRSGDKYCRLPNGCYVYAGR
DGTIAPDGEKGEIVIV GPSVSVGYLGSPELTEKAFTMIDGERAYKTGDAGYVE-NGLLFYNGR
TKKSLGPNRRGEVCVK GPMLMKGYVNNPEATKELIDEE GWLHTGDIGYYDEEKHFFIVDR
_aln.pos 480 490 500 510 520 530 54
4futA G-DLVITGGFNVYP-EIESEIDAMPGWESAVIGVPH ADF-GEGVTAVWRD- -GAT- -I
3c5eA ADDI INSSGYRIGPSEVENALMEHPAWETAVISSPD PVRGEWKAFWLA
3vnqA ADLQIKLRGYRIELSDIETAVRRHDDV-VDAWTVREFKPGD LRLVCAYVAREGSAT
4g36A LKSLIKYKGYQVAPAELESILL DAGVAGLPD DDA-GELPAAVWLE T
2v7bA SDDMLK YVSPVEVEMVLVQHDAVLEAAWGVDH GG- -LVKTRAFWLKREFAP- - S
3f CCA LDFQIKLHGYRMELEEIEHHLRACSYVEGAVIVPIKK- -GEK YDYLLAVWPGEHSFEKE
2dlqA LKSLIKYKGYQVPPAELESVLLQHPSIFDAGVAGVPD PVA-GELPGAVWLESGKNM- -T
_consrvd *
_aln.pos 550 560 570 580 590 600
4futA DE- -AQVLHGLDGQLAFMP VIFVDDLPRNTMGAVQ-NVLRETYDIY
3c5eA HDPEQLTKELQQHVKSV TAPYKYPRKIEFVL-NLPKTVTGKIQRAKLRDKEW-K
3vnqA TARELRNHIKTLLPAYMHPA-RYLPLPGLPRTV GKVDRAAVARSW
4g36A EKEIVDYVAS QVTTAKKLRGGWFVDEVPKGLTGKLDARKIREILIK-
2v7bA EILAEELKAFVKDRLAPHKYPR-DIVFVDDLPKTATGKIQRFKLRE
3f CCA FKLTSAIKKELNERLPNYMIPR-KFMYQSSIPMTPNGKVDRKKLLSEVTA-
2dlqA EKEVMDYVAS QVSNAKRLRGGVRFVDEVPKGLTGKIDGRAIREILK- - consrvd * *
Structural similarity of ANL superfamily members with RpMatB
The RMSD was calculated for the same set of ANL superfamily members as above with RpMatB, using the align command in PyMOL
(htt :/ /www. ymol wi ki . org index, hp/'Aii.gi¾). PyMOL will first do a sequence alignment, followed by a structural superposition, and then carries out cycles of refinement in order to reject structural outliers found during the fit. In the end, the minimized RMSD between the aligned residues using all atoms or backbone atoms is calculated.
Figure imgf000069_0001
Suitably the polypeptide component of the ATP sensor of the invention comprises sequence corresponding to one of the polypeptides in the above table. Although illustrative embodiments of the invention have been disclosed in detail herein, with reference to the accompanying drawings, it is understood that the invention is not limited to the precise embodiment and that various changes and modifications can be effected therein by one skilled in the art without departing from the scope of the invention as defined by the appended claims and their equivalents.
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Kunzelmann, S. and M. R. Webb (2010). "A fluorescent, reagentless biosensor for ADP based on tetramethylrhodamine-labeled ParM." ACS Chem Biol 5(4): 415-425.
Leatherbarrow, R. J. (2009). GraFit Version 7. Horley, UK, Erithacus Software Limited.
Maiti, R., G. H. Van Domselaar, H. Zhang and D. S. Wishart (2004). "SuperPose: a simple server for sophisticated structural superposition." Nucleic Acids Res 32(Web Server issue): W590-594. Okoh, M. P., J. L. Hunter, J. E. Corrie and M. R. Webb (2006). "A biosensor for inorganic phosphate using a rhodamine-labeled phosphate binding protein." Biochemistry 45(49):
14764-14771.
Patergnani, S., F. Baldassari, E. De Marchi, A. Karkucinska-Wieckowska, M. R. Wieckowski and P. Pinton (2014). "Methods to monitor and compare mitochondrial and glycolytic ATP production." Methods Enzymol 542: 313-332.
Selwyn, J. E. and Steinfel.Ji (1972). "Aggregation Equilibria of Xanthene Dyes." Journal of Physical Chemistry 76(5): 762-&.
Vogt, A. D., N. Pozzi, Z. Chen and E. Di Cera (2014). "Essential role of conformational selection in ligand binding." Biophys Chem 186: 13-21.
Wilkins, M. R., E. Gasteiger, A. Bairoch, J. C. Sanchez, K. L. Williams, R. D. Appel and D. F.
Hochstrasser (1999). "Protein identification and analysis tools in the ExPASy server." Methods Mol Biol 112: 531-552.
Zhou, Y., Z. Xu and J. Yoon (2011). "Fluorescent and colorimetric chemosensors for detection of nucleotides, FAD and NADH: highlighted research during 2004-2010." Chem Soc Rev 40(5): 2222-2235.
Exam ple 13 - Additional Mutations
The concentration range of ATP, measurable with a fluorescent reagentless biosensor, an adduct of two tetramethylrhodamines with MatB from Rhodopseudom onas palustris, has been increased. Mutations were introduced into the binding site to modify ATP binding but maintain the concomitant fluorescence signal. Using this signal, the effect was monitored for mutations in different parts of the binding site. Out of these, three variants were characterized, each with a single extra mutation in the phosphate-binding loop. Two variants (T167A and T303A) weakened the binding, changing the dissociation constant from the parent's 6 μΜ to 123 μΜ and 42 μΜ, respectively but having a fluorescence change of ~3-fold on ATP binding. Kinetic measurements showed that the main effect of these mutations was as an increase in dissociation rate constants. These variants widen the range of ATP concentration that can be measured readily by this biosensor to >ιοο μΜ. In contrast, a third variant, S170A, decreases the dissociation constant of ATP to 3.6 μΜ and has a fluorescence change of 4.2 on binding ATP. This variant also shows while decreased ADP affinity, thereby increasing selectivity of ATP over ADP to >200-fold. This had advantages over the parent by increasing sensitivity as well as increasing selectivity during ATP measurement in which ADP is present. Materials. ATP, ADP and GTP were obtained from Sigma-Aldrich. 5- iodoacetamidotetramethylrhodamine (5-IATR) (Corrie and Craik 1994) was a gift from J. Corrie (NIMR, London) or obtained from Anaspec (CA).
Plasm ids. Plasmid pRhoRpMatB (plasmid pTEVs containing the coding sequence of RpMatB (Genbank accession number CAE25665.1) with the point mutations C106A, R286C, Q457C, K488A and an N-terminal His6-tag) was as described, and now termed pTEV5_ RpMatB_i (Vancraenenbroeck and Webb 2015). The QuikChange site- directed mutagenesis protocol (Stratagene) was used for single-site mutations of the pRhoRpMatB plasmid. Plasmids were sequenced (GATC Biotech) to confirm the presence of the mutations. The three variant plasmids, containing additional S170A, T167A or T303A mutations, are termed pTEV5_ RpMatB_2, pTEV5_ RpMatB _3, and pTEV5_ RpMatB_4 respectively.
Preparation of RpMatB variants, labeled with tetram ethylrhodam ine . Protein expression in Escherichia coli, purification and labeling with tetramethylrhodamine were as described (Vancraenenbroeck and Webb 2015). The concentrations of labeled RpMatB variants were determined using the extinction coefficient of the protein at 280 nm, calculated from the sequence via Expasy Protparam (Wilkins, Gasteiger et al. 1999) and the extinction coefficients of tetramethylrhodamine: (31000 M 1 cm 1) and (52000 M 1 cm 1) (Corrie and Craik 1994)
Double labeling was confirmed for the T167A and T303A variants by mass spectrometry. Unlabeled (His6/Cio6A/Ti67A/R286C/Q457C/K488A)RpMatB had a mass of 57304-2 Da. (5-ATR)2-(His6/Cio6A/Ti67A/R286C/Q457C/K488A)RpMatB, had a mass of 58187.3 Da, conforming to the theoretical molecular weight of the unlabeled protein with two 5-ATRs attached (57304.2 + 2 x 441.5). Unlabeled (His6/Cio6A/R286C/T303A/Q457C/K488A)RpMatB had a mass of 57300.9 Da. (5- ATR)2-(His6/Cio6A/Ti67A/R286C/Q457C/K488A)RpMatB, had a mass of 58186.0 Da.
Absorbance and fluorescence m easurem ents. Absorbance was measured on a Jasco V-550 UV-Vis Spectrophotometer. Fluorescent measurements were obtained on a Cary Eclipse spectrofluorometer (Agilent Technologies), using a 3-mm pathlength quartz cuvette (Hellma), unless otherwise mentioned. Excitation and emission slits were set at 5 nm. Protein and nucleotide concentrations and buffer conditions are given in the figure legends. For titrations with Rho-MatB variants, excitation was at 553 nm, emission at 571 nm.
Data from titrations to measure nucleotide binding were corrected for dilution and analyzed with a quadratic binding curve using Grafit software (Leatherbarrow 2009):
Figure imgf000073_0001
where P and L are the total concentrations of protein and ligand, respectively, KA is the dissociation constant, and min and FMAX are the fluorescence intensities of the free and ligand- bound protein, respectively. Stopped-flow m easurem e nts . These were carried out using a HiTech SF-61DX2 apparatus (TgK Scientific, Bradford-upon-Avon, UK) with a xenon-mercury lamp and operated by Kinetic Studio software (TgK Scientific). The excitation wavelength was 545 nm and there was an OG570 cut-off filter (Schott Glass) on the emission. The concentrations in the text and figures are those in the mixing chamber. Data were fitted to theoretical equations using the Kinetic Studio software.
Approach
In addition to two cysteine mutations, the biosensor suitably has a C106A mutation to eliminate background labeling at that cysteine and suitably has a K488A mutation to block the adenylation half-reaction of ATP and malonate to malonyl-AMP and pyrophosphate. The resulting protein adduct, termed Rho-MatB had essentially no enzyme activity, but bound ATP with a ¾ of 6 μΜ. This ATP biosensor, termed Rho- MatB, couples ATP binding to a 3.7-fold increase in fluorescence intensity and measures ATP concentrations in the low micromolar range. Of importance is the fact that this biosensor is greatly selective for ATP over ADP.
We have now developed further variants of Rho-MatB to change the affinity for ATP, while maintaining a significant fluorescence change on ATP binding. In this way the measurable range ATP concentration has been changed. If the affinity is decreased to allow measurements of higher ATP concentrations, a low, sub-stoichiometric concentration of biosensor can be used, minimizing biosensor usage (Solscheid, Kunzelmann et al. 2015). Alternatively, a biosensor for measurements of lower ATP concentrations by increasing the affinity of Rho-MatB for ATP may be increase sensitivity over the parent biosensor.
The method chosen to change affinity was to mutate amino acids identified from the crystal structure as interacting with ATP (Crosby, Rank et al. 2012). Mostly these looked unlikely to affect the lid closure, that is they are not positioned at the hinge or involved in interactions across the cleft, nor might they affect the rhodamines directly. In doing this, there was essentially a survey of the active-site amino acids and the effect of (mainly) alanine mutations on ATP affinity, readily measured using the rhodamine fluorescence signal. This survey resulted in three new variants, one tighter and two weaker in ATP binding, potentially resulting in the ability to measure ATP from sub- micromolar to >ioo μΜ.
Identification of am ino acid residues around MgATP
Amino acid residues were considered that are important for the structural integrity and function of RpMatB. Our first goal was the RpMatB ATP binding site. Residues were chosen that were within 0.6 nm of MgATP as determined using the crystal structure of RpMatB in the MgATP-bound conformation (Crosby, Rank et al. 2012). In addition, sequence homology amongst the ANL family was used (Figure 26 (Figure Si)), so that highly conserved amino acids were avoided as positions to mutate. Some of these residues belong to the A3, A4, A5, A7, A8 and A10 core motifs, which are known to play an important role in adenylate-forming enzymes (Marahiel, Stachelhaus et al. 1997). The positions of these motifs are shown in Figure 21 and details of the motifs are in Figure 26 (Figure Si). The resulting positions that were subsequently mutated successfully are shown in Table C with their motifs along with which part of the ATP structure is closest.
Preparation and testing of variants of Rho-MatB
To examine the effects of substituting these residues on Rho-MatB's function, the individual residues were mutated to alanine on a background of (His6/Cio6A/R286C/Q457C/K488A)RpMatB (Vancraenenbroeck and Webb 2015). In addition two variants had the T167S and T303S mutations, so potentially still allowing binding to the triphosphate of ATP. The mutants were expressed and purified as for the parent RpMatB. Only variants that gave reasonable expression were continued to purification and labeling with 5-iodoactetyltetramethylrhodamine (5-IATR). The labeled proteins were then tested for the fluorescence change upon ATP binding in the presence of Mg2+ (Table C). For variants showing a significant fluorescence change, the affinity for ATP was determined by titration (Table C). None of the variants of Rho- MatB responded to GTP under the same conditions (data not shown).
Table C: Fluorescence change and affinity for ATP binding to variants of Rho-MatB.
Data are from a survey in 50 mM Tris.HCI pH 7.5, 150 mM NaCI, 10 mM MgCI2, 0.3 mg ml"1 bovine serum albumin at 20 °C but without complete optimization of labeling and purification. The motifs are shown in Figure 21. The ratio of fluorescence at saturating ligand to that in the absence of ligand (F+/F.) and the ATP dissociation constant (Kd) were determined by titrations as in Figure 24. The parent Rho-MatB is the protein without any binding site mutations.
Position near Kd for ATP
Variant Motif
ATP Nf- (μΜ)
Parent 2.9 8.8
T163A A3 Y-P 1.8 297 ± 27
S164A A3 a-P 2.7 17.4 ± 0.4
T166A A3 β-Ρ 2.0 156 ± 7
T167A A3 Y-P 2.9 149 ± 7
T167S A3 Y-P 3.3 52 ± 1
R169Ab A3 Y-P 2.8 33 ± 1
S170A A3 Y-P 3.9 4.0 ± 0.1
H207Ab A4 a-P 1.4 224 ± 18
T208A a-P 1.7 52 ± 5
S277A adenine 0.9 3 -
E298A adenine l.l a -
R299A adenine 1.7 2.4 ± 0.1
Y300A A5 adenine 2.1 11.1 ± 0.5
G301A A5 adenine 0.8 a -
M302A A5 ribose 1.5 50 ± 5
T303A A5 a-P 2.6 42 ± 1
T303S A5 a-P 1.7 56 ± 3 I394A adenine 1.1 a
Q490A A1° β-Ρ, γ-Ρ 2.8 66 ± 3 a These ratios are from a single measurement at 750 μΜ ATP. As the fluorescence change was very low, no titration was performed.
b Mutations in the corresponding residue of MatB from Rhizobium trifolii have been studied previously (An, Lee et al. 1999).
The Table shows that the mutations that least perturbed the fluorescence change on ATP binding were all located near the triphosphate. However, the mutations that caused the greatest decrease in affinity (T163A, T166A, T167A, S170A) were all in motif A3, the phosphate binding loop. This suggests that changes to this loop decreases binding but still allows the full conformation change, which controls the interaction between rhodamines and therefore the fluorescence change. In contrast, all the mutations that cause almost complete loss of the fluorescence change are close to the adenosine.
Three variants that retained large changes in fluorescence signals but had potentially useful changes in binding properties (T167A, S170A and T303A) were chosen for further study. They were purified further and their properties were measured and are described in detail.
Weak binding variants, T167A and T303A
Two labeled variants which showed a large fluorescent increase upon MgATP-binding, but also showed a significant reduction in ATP affinity, relative to the parent Rho- RpMatB, were T167A and T303A. Their side chains are involved in hydrogen bonding between RpMatB and the triphosphate of ATP (Figure 21B). Their fluorescence spectra with and without ATP are in Figure 22A and B. The absorbance spectra (Figure 27 (Figure S2A and B)) show the changes on ATP binding typical for tetramethylrhodamine switching between mainly stacked to unstacked configuration. This behaviour was seen previously for the parent Rho-MatB and other proteins labeled with two rhodamines (Chambers, Kajiwara et al. 1974; Hamman, Oleinikov et al. 1996; Okoh, Hunter et al. 2006; Kunzelmann and Webb 2010; Vancraenenbroeck and Webb 2015).
The affinity for ATP and ADP was determined by measuring the fluorescence at different concentrations of the nucleotide (Figure 23A and B). Both show similar fluorescent changes with ATP to the parent Rho-MatB, but the dissociation constants C¾) are much higher (Table D). In contrast, the affinity of ADP was much less affected, remaining very weak (Table D). The change in ATP affinity presumably reflects the importance of hydrogen-bonding interactions of the triphosphate.
Table D: Fluorescence changes, dissociation constants and rate constants for ATP binding to variants of Rho-MatB.
Both the ratio of fluorescence at the saturating ligand to that in the absence of ligand (F+/F-) and the dissociation constants (Kd) were obtained from the data in Figure 23. Association kinetics were from Figure 25: kon is the second order association rate constant, k0n is the dissociation rate constant and k2 is the value obtained for the conformation change. The Kd ratio is for ADP/ATP. Values for the parent biosensor were taken at 25 °C (Vancraenenbroeck and Webb 2015). Note: the protein was purified further than that used in Table C.
Kd (ATP) kon koff k2 Kd (ADP) Kd ratio
Variant F+/F.
(μΜ) foM- 1) (s 1) (s 1) (μΜ)
Parent 3.9 ± 0.2 6.4 ± 0.6 1.8 ± 0.02 8.2 ± 0.2 0.88 ± 0.13 428 ± 50 69
T167A 3.2 ± 0.1 123 ± 4 1.83 ± 0.05 87 ± 4 0.56 ± 0.01 900 ± 28 7.3
T303A 2.7 ± 0.1 42 ± 2 1.60 ± 0.06 16 ± 4 0.60 ± 0.02 1835 ± 114 44
S170A 4.2 ± 0.1 3.8 ± 0.1 1.83 ± 0.10 9 ± 6 0.64 ± 0.02 887 ± 25 233
Calibration curves and effect of ADP
In assays in which ATP is being measured, ADP is likely to be present also and may be at relatively high concentration. In order to show how the presence of ADP affected the response of the variants to ADP, calibration curves were constructed at different levels of ADP (Figure 24). The total concentration of nucleotide (ATP + ADP) was held constant, to mimic partial interconversion of the two nucleotides. In the absence of ADP the response was approximately linear up to a concentration of ATP, 50% of the ¾-value: of course, this is the first part of the binding curve in Figure 23. For both the weak-binding variants there was still a good response with 500 μΜ ADP present, the highest concentration tested. The response was ~50% of that without ADP.
Association kinetics of the weak-binding variants
Although a complete kinetic analysis was beyond the scope of this work, it was important to gauge how the main binding kinetics changed from the parent and so relate that to response time with ATP. Association transients were obtained under pseudo-first order conditions by rapid mixing of each Rho-MatB variant with a large excess of ATP and following fluorescence with time (Figure 25 and Figure 28 (Figure S3)). Qualitatively the weak-binding variants were similar to the parent (Vancraenenbroeck and Webb 2015), showing two distinct phases. The rate constant obtained are in Table D.
The fast phase (Figure 25) had a rate constant that increased linearly with ATP concentration and was interpreted as binding. The association rate constant from the gradient only varied little. The dissociation rate constant could be estimated from the intercept and is the main difference between variants.
The slow phase (Figure 28 (Figure S3)) had a rate constant that did not change significantly with concentration: an average value is shown in Table D. This was interpreted, based on data with the parent Rho-MatB, as a conformation change to the apoprotein prior to binding. This rate constant was very similar to the parent, suggesting that the extra mutations did not affect the conformation change significantly.
Overall, the main effect of the active site mutations is on the dissociation kinetics, which then modify the affinity. Properties of the tight-binding variant, S170 A
In contrast to the two mutations on the phosphate binding loop, described above, the T170A variant strengthens binding. This amino acid does not appear to interact with the triphosphate directly, unlike the hydrogen bonding of T167A and T303A. ATP and ADP binding was measured, as described above for the other variants.
The changes to the fluorescence spectra (Figure 22 C) and absorbance spectra (Figure 27 (Figure S2C)) are similar to those shown above and for the parent Rho-MatB (Vancraenenbroeck and Webb 2015). The fluorescence change is slightly higher than the parent (Table D). Titration with ATP gave a Kd of 3.8 μΜ, approximately two fold tighter than the parent (Figure 23C). However, because ADP binding was apparently weakened, the relative affinity of ATP and ADP is much greater. That can be seen in Figure 23C: at 100 μΜ nucleotide, ATP gives close to maximum fluorescence, ADP shows almost unchanged from the apoprotein. In the calibration curves (Figure 24C), 50 μΜ ADP has no significant effect on the signal with ATP.
Like the two, weak-binding variants, the association kinetics were measured for the S170A Rho-RpMatB (Figure 25) to obtain the second order association rate constant (Table D). This rate constant was very similar for all three variants and the parent Rho- MatB, The second phase, likely to represent a conformation change prior to ATP binding as described above, was also very similar to the other variants and parent rho- MatB (Figure 28 (Figure S3) and Table D). The main difference between these is in the intercept from the fast phase (Figure 25B), representing the dissociation rate constant, which therefore controls the size of the equilibrium dissociation constant.
Conclusions
The mutations in the binding site give us insights into the contributions of specific residues to ATP binding. The fluorescence labelling provides a signal to measures the effect of the amino acid side chain on the affinity of a protein-ligand complex. In particular, the effect of three mutations in the phosphate binding loop were studied in detail, as these gave desirable modifications to the biosensors of the invention for measuring ATP, such as the exemplary Rho-MatB. T167A and T303A decrease the affinity and so provide biosensors in the tens of micro molar ATP, up to >ioo μΜ. These can be used substoichiometrically, for example at <ι μΜ, whereby the concentration of ATP is directly correlated to the fraction of the Rho-MatB in the high fluorescence, ATP-bound state.
In contrast, S170A increased ATP affinity, producing a more sensitive probe for ATP with a larger selectivity for ATP over ADP than the parent biosensor. The greater sensitivity comes from a combination of tighter binding and larger fluorescence enhancement.
REFERENCES TO EXAMPLE 13
An, J. H., G. Y. Lee, et al. (1999). "Identification of residues essential for a two-step reaction by malonyl-CoA synthetase from Rhizobium trifolii." Biochemical Journal 344 : 159-66. Chambers, R. W., T. Kajiwara, et al. (1974). "Effect of dimer formation on the electronic absorption and emission spectra of ionic dyes." Journal of Physical Chemistry 78 : 380- 387.
Corrie, J. E. T. and J. S. Craik (1994). "Synthesis and characterisation of pure isomers of iodoacetamidotetramethylrhodamine." Journal of the Chemical Society. Perkin Transactions I: 2967-2973.
Crosby, H. A., K. C. Rank, et al. (2012). "Structure-guided expansion of the substrate range of methylmalonyl coenzyme A synthetase (MatB) of Rhodopseudomonas palustris." Applied and Environmental Microbiology 78 : 6619-6629.
Finn, R. D., A. Bateman, et al. (2014). "Pfam: the protein families database." Nucleic Acids Research 42 (Database issue): D222-30.
Gulick, A. M. (2009). "Conformational dynamics in the Acyl-CoA synthetases, adenylation domains of non-ribosomal peptide synthetases, and firefly luciferase." ACS Chemical Biology 4 : 811-827. Hamman, B. D., A. V. Oleinikov, et al. (1996). "Tetramethylrhodamine dimer formation as a spectroscopic probe of the conformation of Escherichia coli ribosomal protein L7/L12 dimers." Journal of Biological Chemistry 271fi3): 7568-7573.
Kasha, M. (1963). "Energy transfer mechanisms and the molecular exciton model for molecular aggregates." Radiation Research 20 : 55-70.
Kasha, M., H. R. Rawls, et al. (1965). "The exciton model in molecular spectroscopy." Pure and Applied Chemistry 11: 371-392.
Kunzelmann, S., C. Solscheid, et al. (2014). Fluorescent biosensors: design and application to motility proteins. Fluorescent Methods Applied to Molecular Motors. Experientia Supplementum C. P. Toseland and N. Fili. Basel, Springer. 105 : 25-47.
Kunzelmann, S. and M. R. Webb (2010). "A fluorescent, reagentless biosensor for ADP based on tetramethylrhodamine-labeled ParM." ACS Chemical Biology 5 : 415-425.
Leatherbarrow, R. J. (2009). GraFit Version 7. Horley, U.K., Erithacus Software Ltd.
Marahiel, M. A., T. Stachelhaus, et al. (1997). "Modular peptide synthetases involved in nonribosomal peptide synthesis." Chemical Reviews 97: 2651-2673.
Okoh, M. P., J. L. Hunter, et al. (2006). "A biosensor for inorganic phosphate using a rhodamine-labeled phosphate binding protein." Biochemistry 45 : 14764-14771.
Scholes, G. D. and K. P. Ghiggino (1994). "Electronic interactions and interchromophore electron transfer." Journal of Physical Chemistry 98 : 4580-4590.
Solscheid, C, S. Kunzelmann, et al. (2015). "Development of a reagentless biosensor for inorganic phosphate, applicable over a wide concentration range." Biochemistry 54 : 5054-
5062.
Vancraenenbroeck, R. and M. R. Webb (2015). "A fluorescent, reagentless biosensor for ATP, based on malonyl-coenzyme A synthetase." ACS Chemical Biology 10 : 2650-2657. Wilkins, M. R., E. Gasteiger, et al. (1999). "Protein identification and analysis tools in the ExPASy server." Methods in Molecular Biology 112 : 531-552.
All publications mentioned in the above specification are herein incorporated by reference.

Claims

1. An ATP binding molecule comprising a polypeptide which undergoes a conformational change from a first conformation to a second conformation upon binding of ATP, said polypeptide comprising amino acid sequence corresponding to at least amino acids 5 to 497 of SEQ ID NO: l,
said polypeptide having at least 21% sequence identity to SEQ ID NO: l,
said polypeptide having a value of RMSD <4 A relative to RpMatB in the ATP-bound conformation,
wherein said polypeptide comprises a cysteine residue for attachment of at least one reporter moiety, and comprises at least one reporter moiety attached thereto, wherein said cysteine residue for attachment of at least one reporter moiety is positioned such that said reporter moiety undergoes a change in fluorescence upon changing from said first conformation to said second conformation.
2. An ATP binding molecule according to claim 1 wherein the polypeptide comprises a first cysteine residue at a position corresponding to a position selected from R286 , A282, D283 , H285, E287, S289, A290 , K385 , L383 , G384, 1386 and D287 of SEQ ID NO: 1,
and wherein the polypeptide comprises a second cysteine residue at a position corresponding to a position selected from
Q457, A456, V458 , H460 , G461, G461, Q457, H460 , L462, G464, Q465, K470 , L466, F469 and M471 of SEQ ID NO: 1.
3. An ATP binding molecule according to claim 2 wherein the polypeptide comprises a first cysteine residue at a position corresponding to a position selected from R286 and K385 of SEQ ID NO: 1,
and wherein the polypeptide comprises a second cysteine residue at a position corresponding to a position selected from Q457, G461 and K470 of SEQ ID NO: 1.
4. An ATP binding molecule according to claim 3 wherein the polypeptide comprises a first cysteine residue at a position corresponding to position R286 of SEQ ID NO: 1,
and wherein the polypeptide comprises a second cysteine residue at a position corresponding to position Q457 of SEQ ID NO: 1.
5. An ATP binding molecule according to claim 3 wherein the polypeptide comprises a first cysteine residue at a position corresponding to position K385 of SEQ ID NO: 1,
and wherein the polypeptide comprises a second cysteine residue at a position corresponding to position K470 of SEQ ID NO: 1.
6. An ATP binding molecule according to claim 3 wherein the polypeptide comprises a first cysteine residue at a position corresponding to position R286 of SEQ ID NO: 1,
and wherein the polypeptide comprises a second cysteine residue at a position corresponding to position G461 of SEQ ID NO: 1.
7. An ATP binding molecule according to any of claims 1 to 3, said molecule comprising the amino acid sequence of SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6.
8. An ATP binding molecule according to any preceding claim, said molecule further comprising at least two tetramethylrhodamine moieties attached thereto.
9. An ATP binding molecule according to claim 8 wherein each of said at least two tetramethylrhodamine moieties is independently selected from the group consisting of
5-tetramethylrhodamine and 6-tetramethylrhodamine.
10. An ATP binding molecule according to claim 1 wherein said cysteine residue for attachment of a reporter moiety is at a position corresponding to a position selected from E439 , 1403, P433, G438 , G440 , N492 , K491, V493 , R495, E496 and T497 of SEQ ID NO: 1.
11. An ATP binding molecule according to claim 10 wherein said cysteine residue for attachment of a reporter moiety is at a position corresponding to E439 or N492 of SEQ ID NO: 1.
12. An ATP binding molecule according to any of claims 10 or 11, said molecule comprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:3.
13. An ATP binding molecule according to any of claims 10 to 12, said molecule further comprising at least one diethylaminocoumarin moiety attached thereto.
14. An ATP binding molecule according to claim 13 wherein said diethylaminocoumarin moiety is independently selected from the group consisting of (N-[2-(l-maleimidyl)ethyl]-7-diethylaminocoumarin-3-carboxamide and N-[2- (iodoacetamido)ethyl]-7-diethylaminocoumarin-3-carboxamide).
15. An ATP binding molecule according to any of claims 1 to 14 wherein said polypeptide comprises an amino acid other than Threonine at the position corresponding to T167 of SEQ ID NO: 1.
16. An ATP binding molecule according to claim 15 wherein said polypeptide comprises alanine or serine at the position corresponding to T167 of SEQ ID NO: 1, preferably alanine.
17. An ATP binding molecule according to any of claims 1 to 14 wherein said polypeptide comprises an amino acid other than Serine at the position corresponding to S170 of SEQ ID NO: 1.
18. An ATP binding molecule according to claim 17 wherein said polypeptide comprises alanine at the position corresponding to S170 of SEQ ID NO: 1.
19. An ATP binding molecule according to any of claims 1 to 14 wherein said polypeptide comprises an amino acid other than Threonine at the position corresponding to T303 of SEQ ID NO: 1.
20. An ATP binding molecule according to claim 19 wherein said polypeptide comprises alanine or serine at the position corresponding to T303 of SEQ ID NO: 1, preferably alanine.
21. An ATP binding molecule according to any of claims 1 to 20 wherein said polypeptide comprises an amino acid other than cysteine at the position corresponding to C106 of SEQ ID NO: 1.
22. An ATP binding molecule according to claim 21 wherein said polypeptide comprises alanine at the position corresponding to C106 of SEQ ID NO: 1. ( C106A )
23. An ATP binding molecule according to any of claims 1 to 22 wherein said polypeptide comprises an active site mutation such that the polypeptide is catalytically inactive for ATP hydrolysis.
24. An ATP binding molecule according to claim 23 wherein said active site mutation comprises an amino acid other than lysine at the position corresponding to K488 of SEQ ID NO: 1.
25. An ATP binding molecule according to claim 24 wherein said polypeptide comprises alanine at the position corresponding to K488 of SEQ ID NO: 1. (K488A)
26. An ATP binding molecule according to any of claims 1 to 25 further comprising the sequence of SEQ ID NO: 8.
27. A nucleic acid having a nucleotide sequence encoding the polypeptide portion of an ATP binding molecule according to any of claims 1 to 26.
28. A method for monitoring changes in ATP concentration in a sample comprising contacting said sample with an ATP binding molecule according to any of claims 1 to 20 and determining changes in conformation of said ATP binding molecule, wherein changes in conformation of said ADP binding molecule indicate changes in the concentration of ATP in said sample.
29. A method according to claim 28 wherein the conformation of said ATP binding molecule is monitored by measurement of changes in fluorescence of a fiuorophore comprised by said ATP binding molecule.
30. A method according to claim 28 or claim 29 wherein the sample comprises divalent Magnesium ion (Mg2+).
31. Use of an ATP binding molecule according to any of claims 1 to 26 in the determination of ATP concentration in a sample.
An ATP binding molecule or method substantially as disclosed herein.
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