Nonlinear optical detection of molecules comprising an unnatural amino acid possessing a hyperpolarizability

Abstract

A system for making molecules, and proteins in particular, suitable for detection by a surface-selective nonlinear optical technique. A first use of the invention is for determining a protein's structure in real space and real time. A second use of the invention is to detect a protein or its activity (conformational change). A third use of the invention is for drug screening. A further aspect of the present invention is measuring probe tilt angle orientation in an oriented protein.

1 - 18 . (canceled). 
     
     
         19 . A method for determining if a local conformational change in the structure of a protein occurs upon exposing a protein to a second molecule, the method comprising:
 (a) incorporating an unnatural amino acid into the protein;   (b) chemically coupling the protein of step (a) with a second harmonic (SH)-active or sum frequency (SF)-active moiety that is chemically equipped to react covalently with the unnatural amino acid;   (c) contacting the protein with an interface such that the SH-active or SF-active moiety has a net orientation at the interface;   (d) detecting light emitted using a surface-selective technique; and   (d) measuring a change in one or more physical properties of the emitted light upon exposing the protein to the second molecule, wherein the change in one or more physical properties of the emitted light indicates a local conformational change in the structure of the protein.   
     
     
         20 . The method of  claim 19 , wherein the second molecule is a ligand, a compound, a small molecule, a drug, a peptide, an inhibitor, or a protein. 
     
     
         21 . The method of  claim 19 , further comprising comparing the value of the one or more physical properties measured, with the value of the one or more physical properties measured in the absence of exposure to the second molecule. 
     
     
         22 . The method of  claim 19 , wherein the protein is immobilized on a surface. 
     
     
         23 . The method of  claim 22 , wherein the surface is a glass surface or mica. 
     
     
         24 . The method of  claim 19 , wherein the unnatural amino acid is para-acetyl-phenylalanine (pAcF). 
     
     
         25 . The method of  claim 19 , wherein the protein is an ion channel, an integrin, a kinase, a phosphatase, a receptor, or a G-protein coupled receptor (GPCR). 
     
     
         26 . The method of  claim 25 , wherein the protein is a kinase or a phosphatase. 
     
     
         27 . The method of  claim 26 , wherein a domain of the protein that undergoes a conformational change upon binding to a second molecule is a loop region. 
     
     
         28 . The method of  claim 27 , wherein the loop region is a catalytic loop, a WPD loop, a PTP loop, a recognition loop, or an activation loop. 
     
     
         29 . The method of  claim 19 , wherein the one or more physical properties of the emitted light comprise intensity or polarization dependence. 
     
     
         30 . A method for determining if a local conformational change in the structure of a protein occurs upon exposing the protein to a second molecule, the method comprising:
 (a) incorporating a first unnatural amino acid into the protein which is not SH-active or SF-active;   (b) incorporating a second unnatural amino acid into the protein which is not SH-active or SF-active;   (c) chemically coupling the protein with a second harmonic (SH)-active or sum frequency (SF)-active moiety that is chemically equipped to react covalently with the first unnatural amino acid to create a covalent bond;   (d) chemically coupling the protein in an oriented manner to a surface by reacting the second unnatural amino acid with the surface;   (d) detecting light emitted using a surface-selective technique; and   (e) measuring a change in one or more physical properties of the emitted light upon exposing the protein to the second molecule, wherein the change in one or more physical properties of the emitted light indicates a local conformational change in the structure of the protein.   
     
     
         31 . The method of  claim 30 , wherein the second molecule is a ligand, a compound, a small molecule, a drug, a peptide, an inhibitor, or a protein. 
     
     
         32 . The method of  claim 30 , further comprising comparing the value of the one or more physical properties measured, with the value of the one or more physical properties measured in the absence of exposure to the second molecule. 
     
     
         33 . The method of  claim 30 , wherein the protein is an ion channel, an integrin, a kinase, a phosphatase, a receptor, or a G-protein coupled receptor (GPCR). 
     
     
         34 . The method of  claim 33 , wherein the protein is a kinase or a phosphatase. 
     
     
         35 . The method of  claim 34 , wherein a domain of the protein that undergoes a conformational change upon binding to a second molecule is a loop region. 
     
     
         36 . The method of  claim 35 , wherein the loop region is a catalytic loop, a WPD loop, a PTP loop, a recognition loop, or an activation loop. 
     
     
         37 . The method of  claim 30 , wherein the one or more physical properties of the emitted light comprise intensity or polarization dependence. 
     
     
         38 . The method of  claim 32 , wherein the first unnatural amino acid is para-acetyl-phenylalanine (pAcF).