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Protein Dynamics in Living Cells

Protein Dynamics in Living Cells

Different fluorescence-based techniques are used to study the protein dynamics in living cells. These techniques include FRAP, FRET, and PET.
Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached...

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Related Experiment Video

Updated: Jun 12, 2025

Automated Two-dimensional Spatiotemporal Analysis of Mobile Single-molecule FRET Probes

Automated Two-dimensional Spatiotemporal Analysis of Mobile Single-molecule FRET Probes

Published on: November 23, 2021

Quantitative analysis methods for free diffusion single-molecule FRET experiments.

Irina V Gopich¹, Hoi Sung Chung¹

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

Current Opinion in Structural Biology

|June 10, 2025

Summary

This summary is machine-generated.

Recent advances in quantitative methods enhance single-molecule Förster Resonance Energy Transfer (smFRET) analysis for freely diffusing biomolecules. These improved techniques offer deeper insights into molecular dynamics and biological processes.

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Area of Science:

Biophysics
Biochemistry
Molecular Biology

Background:

Single-molecule Förster Resonance Energy Transfer (smFRET) is crucial for studying biomolecular structure and dynamics.
Analyzing freely diffusing molecules in smFRET presents unique quantitative challenges.

Purpose of the Study:

To review recent advancements in quantitative methods for analyzing freely diffusing molecules in smFRET.
To highlight methods that improve the accuracy and scope of smFRET studies.

Main Methods:

Exploration of traditional FRET efficiency analysis.
Discussion of advanced photon-by-photon techniques, including maximum likelihood estimation.
Introduction of methods that explicitly account for molecular diffusion to correct for biases.

Main Results:

Development of advanced quantitative methods for smFRET data analysis.
Addressing biases related to brightness and diffusivity variations in different molecular states.
Successful application in studying protein folding, DNA dynamics, and protein oligomerization.

Conclusions:

Recent advancements significantly enhance the capabilities of free diffusion-based smFRET.
These tools provide more accurate insights into biomolecular dynamics on biologically relevant timescales.