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Related Concept Videos

Protein Dynamics in Living Cells01:19

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Different fluorescence-based techniques are used to study the protein dynamics in living cells. These techniques include FRAP, FRET, and PET.
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At room temperature, the chair conformer of cyclohexane undergoes rapid ring flipping between two equivalent chair conformers at a rate of approximately 105 times per second. These two chair conformers are in equilibrium. The rapid ring flipping results in the interconversion of the axial proton to an equatorial proton and an equatorial to the axial proton. Such interconversions are too rapid and cannot be detected on the NMR timescale. Hence, the NMR spectrometer cannot distinguish between the...
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Related Experiment Video

Updated: May 27, 2025

Using Three-color Single-molecule FRET to Study the Correlation of Protein Interactions
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Maximum Likelihood Analysis of Diffusing Molecules with Conformational Dynamics in Single-Molecule FRET.

Irina V Gopich1, John M Louis1, Hoi Sung Chung1

  • 1Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, United States.

The Journal of Physical Chemistry. B
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Summary
This summary is machine-generated.

This study introduces an enhanced maximum likelihood analysis (burstML) for single-molecule Förster resonance energy transfer (FRET) experiments, accurately characterizing molecular dynamics and diffusion. The improved method offers robust analysis even with challenging experimental conditions like high noise and varied molecular states.

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Area of Science:

  • Biophysics
  • Single-molecule spectroscopy
  • Protein dynamics

Background:

  • Analyzing single-molecule Förster resonance energy transfer (FRET) data is complex due to molecular transitions, diffusion, and background noise.
  • Existing methods often simplify analysis by excluding translational diffusion, limiting accuracy under non-ideal conditions.

Purpose of the Study:

  • To extend the maximum likelihood analysis of photon bursts (burstML) for comprehensive analysis of single diffusing molecules.
  • To integrate methods for analyzing both conformational dynamics and translational diffusion simultaneously.
  • To provide accurate characterization of molecular parameters under diverse experimental challenges.

Main Methods:

  • Developed an extended burstML approach combining diffusion and conformational dynamics analysis.
  • Integrated existing methods for diffusion-only and dynamics-only analysis.
  • Validated performance on simulated data and compared with the diffusion-excluding colorML method.

Main Results:

  • The enhanced burstML method accurately determines brightness, diffusion time, FRET efficiency, and transition rates.
  • Achieved robust analysis even with fast/slow transitions, high background noise, and unequal state brightness/diffusivity.
  • Demonstrated superior accuracy over colorML when experimental conditions deviate from ideal.

Conclusions:

  • The extended burstML method provides a more comprehensive and accurate analysis of single-molecule FRET data compared to diffusion-excluding methods.
  • This approach is applicable to a wider range of experimental scenarios, including protein folding studies.
  • Offers valuable insights into molecular behavior under challenging conditions.