Nonlinear optical detection of molecules comprising an unnatural amino acid possessing a hyperpolarizability
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. A method for determining if a local conformational change in the structure of a protein domain occurs upon exposing a protein to a second molecule, the method comprising:
(a) genetically incorporating a second harmonic (SH)-active or sum frequency (SF)-active unnatural amino acid into the protein domain to obtain a non-naturally occurring mutant of the protein;
(b) contacting the protein domain with an interface such that the SH-active or SF-active unnatural amino acid has a net orientation at the interface;
(c) 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 domain.
2. The method of claim 1 , wherein the protein is detected in vitro.
3. The method of claim 1 , wherein the protein is detected in vivo.
4. The method of claim 1 , wherein the second molecule is a ligand, a compound, a small molecule, a drug, a peptide, an inhibitor, or a protein.
5. The method of claim 1 , 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.
6. The method of claim 1 , wherein the protein is immobilized on a surface.
7. The method of claim 6 , wherein the surface is a glass surface or mica.
8. The method of claim 1 , wherein the unnatural amino acid is Aladan.
9. The method of claim 1 , wherein the protein is an ion channel, an integrin, a kinase, a phosphatase, a receptor, or a G-protein coupled receptor (GPCR).
10. The method of claim 9 , wherein the protein is a kinase or a phosphatase.
11. The method of claim 10 , wherein the domain of the protein that undergoes a conformational change upon binding to a second molecule is a loop region.
12. The method of claim 11 , wherein the loop region is a catalytic loop, a WPD loop, a PTP loop, a recognition loop, or an activation loop.
13. The method of claim 1 , wherein the one or more physical properties of the emitted light comprise intensity or polarization dependence.
14. A method for determining if a local conformational change in the structure of a protein domain occurs upon exposing a protein to a second molecule, the method comprising:
(a) genetically incorporating a first unnatural amino acid into the protein which is second harmonic (SH)-active or sum frequency (SF)-active to obtain a non-naturally occurring mutant of the protein;
(b) genetically incorporating a second unnatural amino acid into the protein-which is not SH-active or SF-active;
(c) 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 domain of the protein.