METHODS FOR DETECTING ALLOSTERIC MODULATORS OF PROTEIN
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 . A method for identifying an agent B that binds to an allosteric site on a target protein, the method comprising:
a. contacting the target protein with an agent A that binds to an active site of the target protein or wherein agent A is naturally bound to the active site of the target protein when isolated; and
b. contacting the target protein with the agent B, wherein the target protein is labeled with a second harmonic-active moiety having a net orientation at an interface, wherein a detectable signal is generated by the second harmonic-active moiety using a surface selective technique, and wherein the detectable signal indicates a conformational change in the structure of the target protein produced when the agent B binds to an allosteric site on the target protein; and
c. measuring the presence or absence of the detectable signal after the target protein has been contacted with the agent B.
2 . The method of claim 1 , wherein the second harmonic-active moiety is selected from the group consisting of PyMPO maleimide, PyMPO-NHS, PyMPO-succinimidyl ester, Badan, and Acrylodan.
3 . The method of claim 1 , wherein the second harmonic-active moiety is bound to the target protein by one or more sulfhydryl groups on the surface of the target protein.
4 . The method of claim 3 , wherein said one or more sulfhydryl groups are native sulfhydryl groups.
5 . The method of claim 3 , wherein said one or more sulfhydryl groups are engineered sulfhydryl groups.
6 . The method of claim 5 , wherein said one or more sulfhydryl groups are located on target protein amino acid residues known to contact one or more ligand.
7 . The method of claim 2 , wherein the second harmonic-active moiety is PyMPO-maleimide.
8 . The method of claim 2 , wherein the second harmonic-active moiety is bound to the target protein by one or more amine groups on the surface of the target protein.
9 . The method of claim 8 , wherein said one or more amine groups are native amine groups.
10 . The method of claim 8 , wherein said one or more amine groups are engineered amine groups.
11 . The method of claim 10 , wherein said one or more amine groups are located on target protein amino acid residues known to contact one or more one or more ligands.
12 . The method of claim 11 , wherein the second harmonic-active moiety is PyMPO-succinimidyl ester.
13 . The method of claim 12 , wherein the target protein is labeled in situ while bound to a surface.
14 . The method of claim 1 , wherein the second harmonic-active moiety is an unnatural amino acid.
15 . The method of claim 14 , wherein the unnatural amino acid is located in a region of the target protein known to contact one or more ligands.
16 . The method of claim 5 , wherein the unnatural amino acid is Aladan.
17 . The method of claim 6 wherein the ligand is one or more of a protein, a nucleic acid, a phospholipid, a carbohydrate, or a co-factor.
18 . The method of claim 17 , wherein the ligand is a protein member of a kinase signaling cascade.
19 . The method of claim 18 , wherein the target protein is a G protein-coupled receptor, a steroid hormone receptor, or a tyrosine kinase receptor.
20 . The method of claim 19 , wherein the interface is selected from the group consisting of: a glass surface, a polyethylene glycol surface, a supported lipid bilayer surface, a lipid analog bilayer surface, a plastic surface, a metal surface, a latex surface, a rubber surface, a ceramic surface, a polymeric surface, a polypropylene surface, a polyvinylidene difluoride surface, a polyethylene surface.
21 . The method of claim 20 , wherein the surface is derivatized with oligo-PEG molecules or lipids.
22 . The method of claim 21 , wherein the oligo-PEG molecules or lipids are Ni-NTA-bearing oligo-PEG molecules or Ni-NTA-bearing lipids.
23 . The method of claim 20 , wherein the surface is a supported lipid bilayer or a lipid analog bilayer.
24 . The method of claim 23 , wherein the target protein comprises an affinity tag.
25 . The method of claim 24 , wherein the conformational change in the structure of the target protein is detected in real time.
26 . The method of claim 25 , wherein the agent A is a small molecule chemical compound, an antibody, a non-antibody polypeptide, a carbohydrate, an inhibitory nucleic acid, or any combination thereof.
27 . The method of claim 26 , wherein the agent B is a small molecule chemical compound, an antibody, a non-antibody polypeptide, a carbohydrate, an inhibitory nucleic acid, or any combination thereof that specifically binds to the target protein active site.
28 . A method for identifying a site-specific conformational change in the structure of a protein upon binding of an agent to the protein, the method comprising:
a. contacting the target protein with the agent, wherein the target protein is labeled at a first amino acid residue with a second harmonic-active moiety having a net orientation at an interface, wherein a first detectable signal is generated by the second harmonic-active moiety using a surface selective technique, and wherein the first detectable signal indicates a conformational change in the structure of the target protein produced when the agent binds to a site on the target protein;
b. contacting the target protein with the agent, wherein the target protein is labeled at a second amino acid residue with a second harmonic-active moiety having a net orientation at an interface, wherein the second amino acid residue is located in a region of the target protein that differs from the location of the first amino acid residue, wherein a second detectable signal is generated by the second harmonic-active moiety using a surface selective technique, and wherein the second detectable signal indicates a conformational change in the structure of the target protein produced when the agent binds to a site on the target protein; and
c. comparing the first detectable signal with the second detectable signal, wherein a conformational change at the site of the first amino acid residue alone indicates that the agent induces a conformational change to the structure of the protein at the site of the first amino acid residue and wherein a conformational change at the site of the second amino acid residue alone indicates that the agent induces a conformational change to the structure of the protein at the site of the second amino acid residue.
29 . The method of claim 28 , wherein the method includes the initial step of contacting the target protein with an agent that binds to the active site of the target protein.
30 . The method of claim 28 , further comprising repeating steps (b) and (c), wherein one or more additional amino acid residues located at one or more different sites in the target protein are labeled with a second harmonic-active moiety having a net orientation at an interface, wherein one or more additional detectable signals are generated by the second harmonic-active moiety(ies) using a surface selective technique, and wherein the one or more additional detectable signals indicates a conformational change in the structure of the target protein produced when the agent binds to a site on the target protein.
31 - 79 . (canceled)