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Single-Molecule FRET-Resolved Protein Dynamics - from Plasmid to Data in Six Steps
Benjamin Vermeer1, Jannick van Ossenbruggen1, Sonja Schmid2
1Laboratory of Biophysics, Wageningen University & Research, Wageningen, The Netherlands.
Methods in Molecular Biology (Clifton, N.J.)
|October 12, 2023
Summary
This study presents a comprehensive protocol for using single-molecule Förster resonance energy transfer (smFRET) to analyze protein dynamics. The method, demonstrated with heat-shock protein Hsp90, enables detailed investigation of protein conformational changes.
Area of Science:
- Biophysics
- Structural Biology
- Biochemistry
Background:
- Single-molecule Förster resonance energy transfer (smFRET) is crucial for studying biomolecular dynamics.
- Existing smFRET protocols often focus on DNA, limiting protein system analysis.
Purpose of the Study:
- To establish a comprehensive protocol for resolving protein conformational dynamics using smFRET.
- To adapt and demonstrate the smFRET technique for protein systems, starting from plasmid DNA.
Main Methods:
- Detailed protocol for protein production and site-specific bioconjugation.
- Application of total internal reflection fluorescence microscopy (TIRFM) for smFRET measurements.
- Methodology for raw data processing to extract time-resolved dynamics.
Main Results:
- Successful application of smFRET to study the heat-shock protein Hsp90.
- Demonstration of time-resolved protein dynamics analysis.
- Validation of the protocol for protein conformational analysis.
Conclusions:
- The developed smFRET protocol is effective for studying protein conformational dynamics.
- This approach is transferable to a wide range of protein systems.
- Enables detailed exploration of protein conformational energy landscapes.
