SENSOR PROTEINS AND ASSAY METHODS
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.