Methods for determining protein structure using a surface-selective nonlinear optical technique

• US 10,672,502 B2
• Filed: 04/01/2016
• Issued: 06/02/2020
• Est. Priority Date: 04/02/2015
• Status: Active Grant

• Current Assignee: BlueLight Therapeutics, Inc.
• Original Assignee: Biodesy, Inc.
• Inventors: Salafsky, Joshua
• Primary Examiner(s): Borin, Michael L
• Application Number:
  US15/089,013
• Publication Number:
  US 20160292354A1
• Time in Patent Office: 1,523 Days
• Field of Search: None
• CPC Class Codes: G01B 11/14 for measuring distance or clearance between spaced objects or spaced apertures G01B11/26 takes precedence; rangefinders G01C3/00
• CPC Class Codes: G01N 21/21 Polarisation-affecting properties G01N21/19 takes precedence
• CPC Class Codes: G01N 21/636 using an arrangement of pump beam and probe beam; using the measurement of optical non-linear properties; non-linear optics per se G02F1/35
• CPC Class Codes: G01N 2201/06113 Coherent sources; lasers
• CPC Class Codes: G01N 2201/0683 Brewster plate; polarisation controlling elements
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: G01N 33/54373 involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: G01N 33/6803 General methods of protein analysis not limited to specific proteins or families of proteins
• CPC Class Codes: G16B 15/00 ICT specially adapted for analysing two-dimensional or three-dimensional molecular structures, e.g. structural or functional relations or structure alignment

Abstract

Methods, devices, and systems are disclosed for determining protein structure and dynamics using second harmonic generation (SHG) and related surface-selective nonlinear optical techniques.

Claims

1. A method for determining protein structure in solution, the method comprising:

(a) tethering protein molecules to a surface under three or more different sets of experimental conditions known to produce different orientational distributions of the tethered protein molecules, wherein the protein molecules are labeled at one or more known positions with one or more nonlinear-active labels for each experimental condition, and wherein the three or more different sets of experimental conditions comprise two or more of;

(i) tethering the protein molecules using a His-tag attached to the N-terminus, (ii) tethering the protein molecules using a His-tag attached to the C-terminus, and (iii) tethering the protein molecules to at least two surfaces of different composition;

(b) illuminating the tethered protein molecules of step (a) with excitation light of at least one fundamental frequency and a first polarization, wherein the excitation light is provided by at least one light source;

(c) detecting a first intensity of light generated by the one or more nonlinear-active labels as a result of the illumination in step (b) for the three or more different sets of experimental conditions;

(d) illuminating the tethered protein molecules of step (a) with excitation light of the at least one fundamental frequency and a second polarization;

(e) detecting a second intensity of light generated by the one or more nonlinear-active labels as a result of the illumination in step (d) for the three or more different sets of experimental conditions;

(f) calculating intensity ratios for the light detected at the first and second polarizations for each set of experimental conditions to determine a relative orientation of the one or more nonlinear-active labels in the tethered protein molecules for each set of experimental conditions; and

(g) globally fitting data for the relative orientation of the one or more nonlinear-active labels under each set of experimental conditions to a structural model of the protein molecule, wherein the structural model is based on known positions of the one or more nonlinear-active labels within the protein molecule.

2. The method of claim 1, further comprising repeating steps (a) through (f) for at least two different nonlinear-active label-protein conjugates, wherein the nonlinear-active labels are attached to at least two different sites on the protein molecule.

3. The method of claim 2, wherein the at least two different nonlinear-active label-protein conjugates each comprise a single-site cysteine.

4. The method of claim 1, wherein the nonlinear-active labels are nonlinear-active unnatural amino acids.

5. The method of claim 1, wherein the first and the at least second physical properties of light possess the same polarization but are of different magnitudes or intensities.

6. The method of claim 1, wherein the first and at the least second physical properties of light possess different polarizations.

7. The method of claim 4, wherein the nonlinear-active unnatural amino acid is Aladan or a derivative of naphthalene.

8. The method of claim 1, further comprising incorporating x-ray crystallographic data for the protein into the structural model of the protein molecule.

9. The method of claim 1, wherein one of the three or more different sets of experimental conditions comprises applying a first electric field of a first electric field strength to the tethered protein molecules, and another one the three or more different sets of experimental conditions comprises applying a second electric field of a second electric field strength to the tethered protein molecules.

10. The method of claim 9, wherein the first electric field and the second electric field are selected from the group consisting of direct current (DC) fields, alternating current (AC) fields, and any combination thereof.

11. The method of claim 9, wherein the first electric field and the second electric field are applied using an array of electrodes fabricated on the surface.

12. The method of claim 11, wherein the array of electrodes is a circular array as illustrated in FIG. 9.

13. The method of claim 1, wherein one of the three or more different sets of experimental conditions comprises tethering the protein molecules using a first His-tag selected from the group consisting of 2×

His, 4×

His, 6×

His, 8×

His, 10×

His, 12×

His, and 14×

His, and another one of the three or more different sets of experimental conditions comprises tethering the protein molecules using a second His-tag that differs in length from the first His-tag.

14. The method of claim 1, wherein one of the three or more different sets of experimental conditions comprises tethering the protein molecules using a first assay buffer, and another one of the three or more different sets of experimental conditions comprises tethering the protein molecules using a second assay buffer that differs from the first assay buffer.

15. The method of claim 14, wherein the difference between the first assay buffer and the second assay buffer is selected from the group consisting of ionic strength, pH, detergent concentration, calcium ion (Ca²⁺) concentration, magnesium ion (Mg²⁺) concentration, polyethylene glycol concentration, and any combination thereof.

16. The method of claim 1, wherein the difference between one of the three or more different sets of experimental conditions and another one of the three or more different sets of experimental conditions comprises contacting the tethered protein molecules with at least a first ligand that is known to bind to and induce conformational change in the protein molecules.

17. The method of claim 1, wherein the one or more nonlinear-active labels located at the one or more known positions are the same.

19. The method of claim 1, wherein the one or more nonlinear-active labels are selected from the group consisting of second harmonic (SH)-active labels, sum frequency (SF)-active labels, and difference frequency (DF)-active labels.

20. The method of claim 1, wherein the detecting in steps (c) and (e) comprise adjusting the polarization of the light generated by the one or more nonlinear-active labels that reaches a detector.

21. The method of claim 1, wherein the one or more nonlinear-active labels are selected from the group consisting of pyridyloxazole (PyMPO), PyMPO succinimidyl ester, and PyMPO maleimide.

18. The method of 1, wherein the one or more nonlinear-active labels located at the one or more known positions are different.