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 . An apparatus for detection of target protein-allosteric modulator binding interactions, the apparatus comprising:
 a) one or more light sources configured to provide light of one or more fundamental frequencies;   b) a substrate comprising a functionalized surface for attachment of a target protein labeled with a nonlinear-active moiety, wherein the surface-attached target protein has a net orientation at the surface, and wherein the substrate is configured so that surface-attached target protein is illuminated by light of the one or more fundamental frequencies, thereby generating light of a higher order frequency; and   c) a detector for measuring a property of the higher order frequency 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.   
     
     
         81 . The apparatus of  claim 80 , wherein the one or more light sources are one or more lasers. 
     
     
         82 . The apparatus of  claim 80 , wherein the substrate is made of silica, glass, silicon, polystyrene, polycarbonate, nylon, a plastic, a metal, a semiconductor, or an insulator. 
     
     
         83 . The apparatus of  claim 80 , wherein the functionalized surface comprises a supported phospholipid bilayer. 
     
     
         84 . The apparatus of  claim 80 , wherein the detector is a photomultiplier tube (PMT), photodiode, avalanche photodiode, or CCD detector. 
     
     
         85 . The apparatus of  claim 80 , further comprising a monochromator for selection of the fundamental light frequency or higher order light frequency. 
     
     
         86 . The apparatus of  claim 80 , further comprising a bandpass filter, color filter, or interference filter to separate light of the fundamental frequency from light of the higher order frequency. 
     
     
         87 . The apparatus of  claim 80 , further comprising one or more polarizing optics. 
     
     
         88 . The apparatus of  claim 80 , further comprising a computer and software for controlling the illumination of the surface-attached target protein in step (b) and measuring a property of the higher order frequency light, or changes thereof, in step (c) 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. 
     
     
         89 . The apparatus of  claim 80 , 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. 
     
     
         90 . The apparatus of  claim 80 , wherein the nonlinear active moiety is selected from the group consisting of PyMPO maleimide, PyMPO-NHS, PyMPO-succinimidyl ester, Badan, Acrylodan, and an unnatural amino acid. 
     
     
         91 . The apparatus of  claim 80 , wherein the surface-attached target protein is illuminated by the light of the one or more fundamental frequencies using a surface-selective technique. 
     
     
         92 . The apparatus of  claim 81 , wherein the surface selective technique comprises the use of total internal reflection. 
     
     
         93 . The apparatus of  claim 80 , wherein the light of a higher order frequency is second harmonic, sum frequency, or difference frequency light. 
     
     
         94 . The apparatus of  claim 80 , wherein the property of the higher order frequency light to be measured is intensity, wavelength, or polarization. 
     
     
         95 . The apparatus of  claim 80 , wherein the ligand is a protein, a nucleic acid, a phospholipid, a carbohydrate, a small molecule, or a co-factor. 
     
     
         96 . The apparatus of  claim 80 , wherein the allosteric modulator or candidate allosteric modulator is a small molecule, an antibody, a non-antibody polypeptide, a carbohydrate, or an inhibitory nucleic acid. 
     
     
         97 . The apparatus of  claim 80 , wherein the property of the higher order frequency light, or changes thereof, are measured in real-time upon contacting the surface-attached target protein with a ligand known to bind to an active site of the target protein, an allosteric modulator of the target protein, a candidate allosteric modulator of the target protein, or any combination thereof. 
     
     
         98 . The apparatus of  claim 80 , wherein the nonlinear-active moiety is a second harmonic-active moiety, and wherein the light of a higher order frequency is verified as second harmonic light by determining its quadratic dependence on the intensity of the fundamental light.