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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
First Claim
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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.

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