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 comprising:
(a) preparing one or more mutants of a protein by incorporating one or more unnatural amino acid(s) that are second harmonic (SH)-active or sum frequency (SF)-active into the protein;
(b) immobilizing the protein mutants on a surface so as to produce a net orientation of the one or more unnatural amino acid(s) relative to the surface;
(c) measuring a tilt angle of the one or more of the unnatural amino acid(s) relative to the surface in the one or more protein mutants;
(d) determining a structure of the protein using the tilt angle measured in (c).
2. The method of claim 1 , further comprising exposing the one or more mutants of the protein to a second molecule.
3. The method of claim 2 wherein the second molecule is a ligand, a compound, a small molecule, a drug, a peptide, an inhibitor, or a protein.
4. The method of claim 1 wherein the one or more mutant proteins are arrayed on a surface.
5. The method of claim 1 wherein the structure is comprised of real space coordinates of the protein.
6. The method of claim 5 wherein the coordinates are of a particular atom, molecule, group of atoms, or group of protein molecules.
7. The method of claim 1 , wherein the tilt angle is measured in real time.
8. The method of claim 1 , wherein determining the structure of the protein comprises determining a conformational change of the protein.
9. The method of claim 8 , wherein the conformational change is determined in real time and space.
10. The method of claim 9 , wherein the conformational change is determined in real time and space simultaneously.
11. The method of claim 1 , wherein determining the structure of the protein comprises determining structural changes for both buried and surface sites.
12. The method of claim 1 , wherein the tilt angle is an absolute tilt angle relative to the surface.
13. The method of claim 1 , wherein preparing one or more mutants of a protein comprise genetically incorporating a series of unnatural amino acids into different mutants of the protein.
14. The method of claim 1 , further comprising determining a three-dimensional structure of the protein.
15. The method of claim 1 , further comprising comparing the determined structure with a X-ray crystal structure of the protein.
16. The method of claim 1 , wherein the unnatural amino acid is Aladan.
17. The method of claim 1 , wherein the at least one of the one or more unnatural amino acid(s) are incorporated into a buried residue of the protein.
18. The method of claim 1 , wherein the one or more unnatural amino acid(s) are incorporated into the protein site-specifically.
19. The method of claim 1 , further comprising incorporation of a different unnatural amino acid into the protein.
20. The method of claim 19 , wherein the different unnatural amino acid is not second-harmonic active.