Methods for detecting allosteric modulators of protein

Abstract

The present invention discloses, inter alia, methods for labeling a target protein with an SHG-active probe for detection by second harmonic or sum-frequency generation in order to identify agents which bind to an allosteric site on the target protein thereby altering its structural conformation

1 - 79 . (canceled) 
     
     
         80 . A kit comprising:
 one or more of:   a. one or more SHG or SFG-active labels for conjugation to a target protein;   b. one or more reagents for labeling proteins with SHG or SFG-active labels;   c. one or more surfaces for binding a target protein;   d. one or more co-factors necessary for target protein functionality;   e. one or more peptides and/or proteins known to bind to one or more target proteins;   f. one or more antibodies known to bind to one or more target proteins; and   g. one or more components for introducing SHG- or SFG-active unnatural amino acid (UAA) labels into proteins; and   one or more signal peripherals for use with an apparatus for detection of target protein-allosteric modulator interactions.   
     
     
         81 . The kit of  claim 80 , wherein the one or more SHG or SFG-active labels are selected from the group consisting of maleimide labels, PyMPO-NHS, oxazole labels, BADAN (6-Bromoacetyl-2-Dimethylaminonaphthalene), ACRYLODAN™ (6-Acryloyl-2-Dimethylaminonaphthalene), and coumarin-based dyes. 
     
     
         82 . The kit of  claim 80 , wherein the surface for binding a target protein is selected from the group consisting of glass, plastic, metal, latex, rubber, ceramic, and polymer. 
     
     
         83 . The kit of  claim 81 , wherein the polymer is polypropylene, polyvinylidene difluoride, polystyrene, or polyethylene. 
     
     
         84 . The kit of  claim 80 , wherein the surface for binding a target protein comprises at least one preformed surface feature. 
     
     
         85 . The kit of  claim 84 , wherein the at least one preformed surface feature is a trench, a v-groove, a mesa structure, and/or a well. 
     
     
         86 . The kit of  claim 80 , wherein the surface for binding a target protein is pre-coated with one or more molecules which provide a surface chemistry suitable for attachment to a target protein. 
     
     
         87 . The kit of  claim 86 , wherein the one or more molecules are aldehyde silanes, BSA-NHS, or oligo-polyethylene glycol. 
     
     
         88 . The kit of  claim 87 , wherein the oligo-polyethylene glycol is nickel-oligo-polyethylene glycol. 
     
     
         89 . The kit of  claim 80 , wherein the surface for binding a target protein is pre-coated with a supported lipid bilayer or supported lipid analog bilayer. 
     
     
         90 . The kit of  claim 80 , wherein the signal peripheral for use with an apparatus for detection of target protein-allosteric modulator interactions is a pass-filter, a color filter, an interference filter, a polarizing optic, or one or more mirrors or lenses for directing and focusing a beam. 
     
     
         91 . The kit of  claim 80 , wherein the co-factor necessary for target protein functionality is thiamine pyrophosphate, NAD + , NADP + , pyridoxal phosphate, lipoamide, methylcobalamin, cobalamine, biotin, Coenzyme A, tetrahydrofolic acid, menaquinone, ascorbic acid, flavin mononucleotide, flavin adenine dinucleotide, Coenzyme F420, adenosine triphosphate (ATP), guanosine triphosphate (GTP), S-adenosyl methionine, Coenzyme B Coenzyme M, Coenzyme Q, cytidine triphosphate, glutathione, heme, nucleotide sugars, pyrroloquinoline, quinone, or tetrahydrobiopterin. 
     
     
         92 . A system for using a non-linear optical technique to screen for and identify agents which bind to and modulate allosteric sites on target proteins, the system comprising:
 a. a target protein labeled with a non-linear-active moiety; and   b. an apparatus for detection of target protein-agent interactions, the apparatus comprising i) a source of fundamental light; ii) a substrate comprising the target protein labeled with a non-linear-active moiety, and iii) a detector for measuring a property of light generated by a nonlinear transformation of the fundamental light,   
       wherein binding of the agent to an allosteric site on the target protein results in a change in the property of the light generated by the non-linear transformation and is detected by the apparatus. 
     
     
         93 - 107 . (canceled) 
     
     
         108 . The system of  claim 92 , wherein the non-linear transformation comprises second harmonic generation (SHG), sum frequency generation (SFG), or difference frequency generation (DFG). 
     
     
         109 . The system of  claim 92 , wherein the non-linear-active moiety comprises a second harmonic-active, sum frequency-active, or difference frequency-active moiety. 
     
     
         110 . The system of  claim 92 , wherein the target protein is selected from the group consisting of a G protein-coupled receptor, a steroid hormone receptor, or a tyrosine kinase receptor. 
     
     
         111 . The system of  claim 92 , wherein the non-linear active moiety is selected from the group consisting of PyMPO maleimide, PyMPO-NHS, PyMPO-succinimidyl ester, Badan, and Acrylodan. 
     
     
         112 . The system of  claim 92 , wherein the source of the fundamental light is a laser. 
     
     
         113 . The system of  claim 92 , wherein the substrate is made of silica, glass, silicon, polystyrene, polycarbonate, nylon, a plastic, a metal, a semiconductor, or an insulator. 
     
     
         114 . The system of  claim 92 , wherein the substrate further comprises a supported lipid phospholipid bilayer. 
     
     
         115 . The system of  claim 92 , wherein the detector is a photomultiplier tube (PMT), photodiode, avalanche photodiode, or CCD detector. 
     
     
         116 . The system of  claim 92 , wherein the apparatus further comprises a computer and software for controlling illumination of the target protein with the fundamental light and measuring the property of the light generated by a nonlinear transformation of the fundamental light, or changes thereof, upon contacting the surface-attached target protein with a ligand known to bind to an active site of the target protein, and (i) an allosteric modulator of the target protein, or (ii) a candidate allosteric modulator of the target protein. 
     
     
         117 . The system of  claim 92 , wherein the property of the light generated by the non-linear transformation is intensity, wavelength, or polarization. 
     
     
         118 . The system of  claim 92 , wherein the agent is a small molecule, an antibody, a non-antibody polypeptide, a carbohydrate, or an inhibitory nucleic acid.