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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: Sep 17, 2025

Making Precise and Accurate Single-Molecule FRET Measurements using the Open-Source smfBox

Making Precise and Accurate Single-Molecule FRET Measurements using the Open-Source smfBox

Published on: July 5, 2021

Model-free photon analysis of diffusion-based single-molecule FRET experiments.

Ivan Terterov¹, Daniel Nettels², Tanya Lastiza-Male^3,4

¹Department of Chemical and Structural Biology, Weizmann Institute of Science, Rehovot, Israel. ivan.terterov@weizmann.ac.il.

Nature Communications

|July 2, 2025

Summary

This summary is machine-generated.

Analyzing single-molecule Förster resonance energy transfer (smFRET) data, this study introduces a model-free method. It accurately quantifies protein dynamics across various timescales, validating current analysis techniques.

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Last Updated: Sep 17, 2025

Making Precise and Accurate Single-Molecule FRET Measurements using the Open-Source smfBox

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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

Area of Science:

Biophysics
Protein Dynamics
Single-Molecule Biophysics

Background:

Diffusion-based single-molecule Förster resonance energy transfer (smFRET) experiments commonly use Markov models to analyze protein dynamics.
FRET efficiencies are projections of conformational space, potentially leading to observed non-Markovian dynamics.
Model-free quantification of FRET efficiency fluctuations is needed.

Purpose of the Study:

To develop and present a model-free approach for quantifying FRET efficiency fluctuations.
To overcome artifacts from finite photon trajectory lengths and confocal volume diffusion.
To rigorously validate current model-based analysis methods.

Main Methods:

Determination of FRET efficiency correlation functions.
Artifact removal related to photon trajectory length and molecular diffusion.
Application to both simulated and experimental smFRET data.

Main Results:

The developed method accurately quantifies protein dynamics over nano- to millisecond timescales.
Correlation functions are free from common experimental artifacts.
The approach provides robust validation for existing Markov model analyses.

Conclusions:

A novel model-free method for analyzing smFRET data is presented.
This approach accurately captures protein dynamics across a wide range of timescales.
The findings support and validate current model-based approaches in smFRET analysis.