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Fast three-color single-molecule FRET using statistical inference.

Janghyun Yoo1, Jae-Yeol Kim1, John M Louis1

  • 1Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, 20892-0520, USA.

Nature Communications
|July 5, 2020
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Summary
This summary is machine-generated.

This study introduces a novel three-color Förster resonance energy transfer (FRET) spectroscopy method for analyzing protein dynamics. The technique enhances time resolution and reduces photobleaching for disordered protein studies.

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Area of Science:

  • Biophysics
  • Spectroscopy
  • Protein Dynamics

Background:

  • Förster resonance energy transfer (FRET) spectroscopy is vital for studying molecular dynamics.
  • Traditional multi-color FRET methods face challenges like acceptor photobleaching, limiting time resolution.
  • Disordered proteins exhibit complex conformational dynamics crucial for biological function.

Purpose of the Study:

  • To develop an advanced three-color FRET spectroscopy technique for sub-millisecond conformational dynamics.
  • To overcome photophysical limitations in FRET experiments, particularly acceptor photobleaching.
  • To enable precise analysis of protein folding and binding in disordered proteins.

Main Methods:

  • A novel scheme using single continuous-wave laser excitation of the donor.
  • Simultaneous collection of two-color and three-color FRET data by exploiting molecular species with absent/photobleached acceptors.
  • Global maximum likelihood analysis of photon trajectories for parameter determination.
  • Co-parallelization of CPU-GPU processing for efficient likelihood calculations.

Main Results:

  • Successful determination of three FRET efficiencies and kinetic parameters without alternating excitation.
  • Demonstrated alleviation of acceptor photobleaching, enabling high time-resolution experiments.
  • Significant reduction in likelihood calculation time through optimized processing.

Conclusions:

  • The developed three-color FRET method offers enhanced capabilities for probing sub-millisecond protein dynamics.
  • This approach provides a robust tool for studying disordered proteins, protein folding, and binding events.
  • The computational efficiency achieved allows for more accessible and detailed kinetic analyses.