Biophysics and Physicobiology
Online ISSN : 2189-4779
ISSN-L : 2189-4779
Regular Article
Second harmonic generation polarization microscopy as a tool for protein structure analysis
Junichi KaneshiroYasushi OkadaTomohiro ShimaMika TsujiiKatsumi ImadaTaro IchimuraTomonobu M. Watanabe
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Supplementary material

2019 Volume 16 Pages 147-157

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Abstract

Cryo-electron microscopy and X-ray crystallography have been the major tools of protein structure analysis for decades and will certainly continue to be essential in the future. Moreover, nuclear magnetic resonance or Förster resonance energy transfer can measure structural dynamics. Here, we propose to add optical second-harmonic generation (SHG), which is a nonlinear optical scattering process sensitive to molecular structures in illuminated materials, to the tool-kit of structural analysis methodologies. SHG can be expected to probe the structural changes of proteins in the physiological condition, and thus link protein structure and biological function. We demonstrate that a conformational change as well as its dynamics in protein macromolecular assemblies can be detected by means of SHG polarization measurement. To prove the capability of SHG polarization measurement with regard to protein structure analysis, we developed an SHG polarization microscope to analyze microtubules in solution. The difference in conformation between microtubules with different binding molecules was successfully observed as polarization dependence of SHG intensity. We also succeeded in capturing the temporal variation of structure in a photo-switchable protein crystal in both activation and inactivation processes. These results illustrate the potential of this method for protein structure analysis in physiological solutions at room temperature without any labeling.

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© 2019 THE BIOPHYSICAL SOCIETY OF JAPAN
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