WO2016156809A1 - Capteur de l'atp - Google Patents

Capteur de l'atp 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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PCT/GB2016/050818
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English (en)
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Martin R. WEBB
Renee VANCRAENENBROECK
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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

L'invention concerne une molécule de liaison à l'ATP comprenant un polypeptide qui subit une modification conformationnelle, d'une première conformation en un deuxième conformation lors de la liaison d'ATP, ledit polypeptide comprenant la séquence d'acides aminés correspondant au moins aux acides aminés 5 à 497 de la séquence SEQ ID NO : 1, ledit polypeptide présentant au moins 21 % d'identité de séquence avec la séquence SEQ ID NO : 1, ledit polypeptide présentant une valeur d'écart-type < 4 Å par rapport à RpMat B dans la conformation liée à l'ATP, ledit polypeptide comprenant un résidu de cystéine pour la fixation d'au moins un fragment rapporteur et comprenant au moins un fragment rapporteur fixé à celui-ci, ledit résidu de cystéine pour la fixation d'au moins un fragment rapporteur étant positionné de telle sorte que le dit fragment rapporteur subit un changement de fluorescence lors du changement de ladite première conformation en ladite deuxième conformation. L'invention concerne également l'utilisation d'un tel capteur et des procédés pour l'analyse de l'ATP.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000068418A1 (fr) * 1999-05-10 2000-11-16 Medical Research Council Dosages des nucleosides diphosphates et des triphosphates
WO2010032001A1 (fr) * 2008-09-19 2010-03-25 Medical Research Council Capteur

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000068418A1 (fr) * 1999-05-10 2000-11-16 Medical Research Council Dosages des nucleosides diphosphates et des triphosphates
WO2010032001A1 (fr) * 2008-09-19 2010-03-25 Medical Research Council Capteur

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CROSBY H A ET AL: "Structure-guided expansion of the substrate range of methylmalonyl coenzyme a synthetase (MatB) of rhodopseudomonas palustris", APPLIED AND ENVIRONMENTAL MICROBIOLOGY SEPTEMBER 2012 AMERICAN SOCIETY FOR MICROBIOLOGY USA, vol. 78, no. 18, September 2012 (2012-09-01), pages 6619 - 6629, XP002759245, DOI: 10.1128/AEM.01733-12 *
IMAMURA HIROMI ET AL: "Visualization of ATP levels inside single living cells with fluorescence resonance energy transfer-based genetically encoded indicators", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, NATIONAL ACADEMY OF SCIENCES, US, vol. 106, no. 37, 15 September 2009 (2009-09-15), pages 15651 - 15656, XP002575707, ISSN: 0027-8424, [retrieved on 20090831], DOI: 10.1073/PNAS.0904764106 *
KORYAKINA IRINA ET AL: "Mutant Malonyl-CoA Synthetases with Altered Specificity for Polyketide Synthase Extender Unit Generation", CHEMBIOCHEM, vol. 12, no. 15, October 2011 (2011-10-01), pages 2289 - 2293, XP002759244 *
VANCRAENENBROECK RENEE ET AL: "A Fluorescent, Reagent less Biosensor for ATP, Based on Malonyl-Coenzyme A Synthetase", ACS CHEMICAL BIOLOGY, vol. 10, no. 11, November 2015 (2015-11-01), pages 2650 - 2657, XP002759246 *

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