Sensor proteins and assay methods

Abstract

The present invention relates to biosensors. In some embodiments, the biosensors are modified ligand binding molecules. In some embodiments, the modified ligand binding molecule is a phosphate binding protein (PBP). In some embodiments, the modified ligand binding molecules are labeled to be capable of RET, e.g., comprising a donor and acceptor moiety. In some embodiments of the invention, there is a detectable change in RET (e.g., FRET) when the modified ligand binding molecule binds and/or releases the ligand (e.g., phosphate). The invention also provides related methods, reactions and assays.

1 . A phosphate binding protein comprising a resonance energy transfer (RET) pair of moieties comprised of at least one donor moiety and at least one acceptor moiety, wherein the phosphate binding protein is capable of binding a phosphate and wherein the binding results in a change in RET. 
     
     
         2 . The protein of  claim 1 , wherein RET increases. 
     
     
         3 . The protein of  claim 1 , wherein RET decreases. 
     
     
         4 . The protein of  claim 1 , wherein the phosphate is inorganic phosphate (Pi). 
     
     
         5 . The protein of  claim 1 , wherein the change in RET is caused by a conformational change of the protein upon binding the phosphate. 
     
     
         6 . The protein of  claim 1 , wherein the change in RET is caused by a conformational change of the protein upon releasing the phosphate. 
     
     
         7 . The protein of  claim 1 , wherein the distance between the at least two moieties is altered upon binding the phosphate. 
     
     
         8 . The protein of  claim 1 , wherein the orientation between the at least two moieties is altered upon binding the phosphate. 
     
     
         9 . The protein of  claim 1 , wherein the RET pair is capable of time resolved RET. 
     
     
         10 . The protein of  claim 1 , wherein the at least one acceptor moiety is selected from the group consisting of a fluorescein, a rhodamine, a GFP, a GFP derivatives, a fluorescent protein, a FITC, a 5-carboxyfluorescein, a 6-carboxyfluorescein, a 7-hydroxycoumarin-3-carboxamide, a 6-chloro-7-hydroxycoumarin-3-carboxamide, a fluorescein-5-isothiocyanate, a gdichlorotriazinylaminofluorescein, a tetramethylrhodamine-5-isothiocyanate, tetramethylrhodamine-6-isothiocyanate, a succinimidyl ester of 5-carboxyfluorescein, a succinimidyl ester of 6-carboxyfluorescein, a 5-carboxytetramethylrhodamine, a 6-carboxymethylrhodamine, a 7-amino-4-methylcoumarin-3-acetic acid, Alexa Fluor 488, Alexa Fluor 633, Alexa Fluor 647, 6-IAF, 5-IAF, BODIPY FL maleimide, BODIPY FL iodoacetamide, fluorescein-5-maleimide, Oregon Green 488 iodoacetamide, Oregon Green 488 maleimide and 5-(bromomethyl)fluorescein. 
     
     
         11 . The protein of  claim 1 , wherein the donor moiety comprises a luminescent metal complex. 
     
     
         12 . The protein of  claim 11 , wherein the luminescent metal complex comprises an organic antenna moiety, a metal liganding moiety and a lanthanide metal ion. 
     
     
         13 . The protein of  claim 12 , wherein the luminescent metal complex is a lanthanide metal complex. 
     
     
         14 . The protein of  claim 13 , wherein the lanthanide metal complex comprises an organic antenna moiety, a metal liganding moiety and a lanthanide metal ion. 
     
     
         15 . The protein of  claim 14 , wherein the lanthanide metal ion is selected from the group consisting of: Sm(M), Ru(III), Eu (III), Gd(III), Tb(III), and Dy(III). 
     
     
         16 . The protein of  claim 14 , wherein the lanthanide ion is a Europium ion. 
     
     
         17 . The protein of  claim 14 , wherein the lanthanide ion is a Terbium ion. 
     
     
         18 . The protein of  claim 14 , wherein the organic antenna moiety is selected from the group consisting of: rhodamine 560, fluorescein 575, fluorescein 590, 2-quinolone, 4-quinolone, 4-trifluoromethylcoumarin (TFC), 7-diethyl-amino-coumarin-3-carbohydrazide, 7-amino-4-methyl-2-coumarin (carbostyril 124), 7-amino-4-methyl-2-coumarin (coumarin 120), 7-amino-4-trifluoromethyl-2-coumarin (coumarin 124), and aminomethyltrimethylpsoralen. 
     
     
         19 . The protein of  claim 14 , wherein the metal liganding moiety is a metal chelating moiety selected from the group consisting of: EDTA, DTPA, TTHA, DOTA, NTA, HDTA, DTPP, EDTP, HDTP, NTP, DOTP, DO3A, DOTAGA, and NOTA. 
     
     
         20 . The protein of  claim 13 , wherein the lanthanide metal complex has a structure:
   -L n -A-S n -C M ,   
       or
   -L n -C M -S n -A, 
 
       wherein A represents an organic antenna moiety;
 L represents a linker; 
 S represents a spacer; 
 n can be 0 or 1; 
 C represents a metal chelating moiety; and 
 M represents a lanthanide metal ion coordinated to C. 
 
     
     
         21 . The protein of  claim 11 , wherein the luminescent metal complex comprises CS124-DTPA-Phe-NCS-Tb or CS124-DTPA-EMCH-Th. 
     
     
         22 . The protein of  claim 1 , wherein the protein has at least one non-native cysteine amino acid. 
     
     
         23 . The protein of  claim 22 , wherein the first or second moiety is attached to the non-native cysteine amino acid. 
     
     
         24 . The protein of  claim 1 , wherein the protein has at least two non-native cysteine amino acids. 
     
     
         25 . The protein of  claim 24 , wherein the first and second moieties are attached to the non-native cysteine amino acids. 
     
     
         26 . The protein of  claim 22 , wherein the at least one non-native cysteine amino acids is introduced by substituting or inserting the cysteine amino acid into the protein. 
     
     
         27 . The protein of  claim 1 , wherein the amino acid sequence of the PBP is derived from the phoS gene. 
     
     
         28 . The protein of  claim 27 , wherein the amino acid sequence encoded by the phoS gene is SEQ ID NO: 1 or SEQ ID NO:2. 
     
     
         29 . The protein of  claim 27 , wherein the protein has at least one non-native cysteine amino acid. 
     
     
         30 . The protein of  claim 27 , comprising an amino acid substitution selected from the group consisting of A47C, A197C, Q201C and E268C. 
     
     
         31 . The protein of  claim 27 , wherein the protein has at least two non-native cysteine amino acids. 
     
     
         32 . The protein of  claim 31 , comprising an amino acid substitution selected from the group consisting of A47C, A197C, Q201C and E268C. 
     
     
         33 . The protein of  claim 31 , comprising at least 2 amino acid substitutions selected from the group consisting of A197C/E268C, A47C/A197C, A47C/E268C, Q201C/E268C, A47C/Q201C and A 197C/Q201C. 
     
     
         34 . The protein of  claim 27 , wherein the first or second moiety is attached to a non-native cysteine amino acid. 
     
     
         35 . The protein of  claim 27 , wherein the first and second moieties are attached to non-native cysteine amino acids. 
     
     
         36 . The protein of  claim 1 , wherein the phosphate binding protein comprises an amino acid sequence 90% homologous to SEQ ID NO: 1 or SEQ ID NO:2. 
     
     
         37 . The protein of  claim 36 , wherein the phosphate binding protein comprises at least one non-native cysteine amino acid. 
     
     
         38 . The protein of  claim 1 , wherein the at least one donor moiety is linked to the phosphate binding protein via an amine or thiol linkage. 
     
     
         39 . The protein of  claim 1 , wherein the at least one acceptor moiety is linked to the phosphate binding protein via an amine or thiol linkage. 
     
     
         40 . A method of measuring phosphate in a first sample comprising:
 (a) contacting the first sample with a protein of  claim 1 ;   (b) exposing (a) to a wavelength of light that excites the donor moiety of the RET pair; and   (c) measuring the emission from the acceptor moiety of the RET pair.   
     
     
         41 . The method of  claim 40 , comprising measuring the emission from the donor moiety of the RET pair. 
     
     
         42 . The method of claim. 41, comprising calculating a ratio between the emission of the donor and acceptor moieties of the RET pair. 
     
     
         43 . The method of  claim 40 , further comprising:
 (i) contacting a second sample with a protein of  claim 1 , wherein the second sample comprises a known amount of the phosphate;   (ii) exposing (i) to a wavelength of light that excites the donor moiety of the RET pair; and   (iii) measuring the emission from the acceptor moiety of the RET pair.   
     
     
         44 . The method of  claim 43 , comprising measuring the emission from the donor moiety of the RET pair in (ii). 
     
     
         45 . The method of  claim 44 , comprising calculating a ratio between the emission of the donor and acceptor moieties of the RET pair in (ii). 
     
     
         46 . The method of  claim 40 , further comprising:
 (i) separately contacting multiple samples with a protein of  claim 1 , wherein the multiple samples comprise a known amount of the phosphate;   (ii) exposing (i) to a wavelength of light that excites the donor moiety of the RET pair; and   (iii) measuring the emission from the acceptor moiety of the RET pair in each sample.   
     
     
         47 . The method of  claim 46 , wherein the amount of phosphate in the first sample is determined by comparing the emission from the first sample to the multiple samples. 
     
     
         48 . The method of  claim 47 , comprising measuring the emission from the donor moiety of the RET pair in (iii). 
     
     
         49 . The method of  claim 48 , comprising calculating a ratio between the emission of the donor and acceptor moieties of the RET pair in (iii). 
     
     
         50 . The method of  claim 40 , wherein measuring the emission occurs at multiple time points. 
     
     
         51 . A method for measuring phosphodiesterase activity of a compound comprising:
 a) contacting the compound and a phosphodiesterase substrate (e.g., cAMP),   b) contacting (a) with a phosphatase capable of removing a phosphate that is no longer part of a phosphodiester bond on the substrate;   c) contacting (b) with a modified PBP; and   d) measuring fluorescence.   
     
     
         52 . The method of  claim 51 , wherein the modified PBP comprises one fluorescent label, wherein the fluorescence of the PBP differ when bound to phosphate as compared to when it is not bound to phosphate. 
     
     
         53 . A method for measuring phosphodiesterase activity of a compound comprising:
 a) contacting the compound and a phosphodiesterase substrate (e.g., cAMP),   b) contacting (a) with a phosphatase capable of removing a phosphate that is no longer part of a phosphodiester bond on the substrate;   c) contacting (b) with the phosphate binding protein of  claims 1 ; and   d) measuring RET.   
     
     
         54 . The method of  claim 53 , wherein (c) is exposed to a wavelength or wavelengths of light that excite the donor moiety. 
     
     
         55 . The method of  claim 53 , wherein (a), (b), and (c) are carried out simultaneously. 
     
     
         56 . The method of  claim 53 , wherein measuring RET is done in real time or as kinetic measurements. 
     
     
         57 . The method of  claim 52 , wherein (a), (b), (c) or any combination thereof comprises a phosphate mop. 
     
     
         58 . The method of  claim 52 , wherein (a) comprises a potential modulator of the phosphodiesterase activity of the compound. 
     
     
         59 . The method of  claim 53 , wherein RET is measured in (a), (b) or (a) and (b). 
     
     
         60 . The method of  claim 52 , further comprising control reactions. 
     
     
         61 . A method for measuring kinase activity of a compound comprising:
 a) contacting the compound and a phosphorylation substrate for the kinase activity,   b) contacting (a) with a phosphatase capable of removing a phosphate added by the kinase activity of the compound;   c) contacting (b) with a modified PBP comprising a RET pair; and   d) measuring RET.   
     
     
         62 . The method of  claim 61 , wherein (c) is exposed to a wavelength or wavelengths of light that excite the donor moiety. 
     
     
         63 . The method of  claim 61 , wherein (a), (b), and (c) are carried out simultaneously. 
     
     
         64 . The method of  claim 61 , wherein RET is measured in real time or as kinetic measurements. 
     
     
         65 . The method of  claim 61 , wherein (a), (b), (c) or any combination thereof comprises a phosphate mop. 
     
     
         66 . The method of  claim 61 , wherein (a) comprises a potential modulator of the kinase activity of the compound 
     
     
         67 . The method of  claim 61 , wherein RET is measured in (a), (b) or (a) and (b). 
     
     
         68 . The method of  claim 61 , further comprising control reactions. 
     
     
         69 . A method for measuring kinase activity of a compound comprising:
 a) preparing a solution comprising the compound, a phosphorylation substrate for the kinase activity, a phosphatase capable of removing a phosphate added by the kinase activity of the compound, and a modified PBP comprising a RET pair; and   b) measuring RET.   
     
     
         70 . The method of  claim 69 , wherein RET is measured in real time or as kinetic measurements. 
     
     
         71 . The method of  claim 69 , wherein (a) comprises a phosphate mop. 
     
     
         72 . The method of  claim 69 , further comprising control reactions.