Abstract
Single-molecule Förster resonance energy transfer (smFRET) is a powerful technique for the detection of conformational dynamics of biomolecules. While many smFRET experiments are performed using dye-labeled DNA, here we describe a comprehensive protocol to resolve the conformational dynamics of a protein system – notably from plasmid to data. Using the example of the heat-shock protein Hsp90, we describe the protein production and threefold site-specific bioconjugation, the smFRET measurement using total internal reflection fluorescence microscopy (TIRFM), and raw data processing to reveal time-resolved protein dynamics. The described smFRET approach is readily transferrable to the study of many more all-protein systems and their conformational energy landscape.
Original language | English |
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Title of host publication | Methods in Molecular Biology |
Publisher | Humana Press |
Pages | 267-291 |
Number of pages | 25 |
Volume | 2694 |
ISBN (Electronic) | 9781071633779 |
ISBN (Print) | 9781071633762 |
DOIs | |
Publication status | Published - 2024 |
Publication series
Name | Methods in Molecular Biology |
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Volume | 2694 |
ISSN (Print) | 1064-3745 |
ISSN (Electronic) | 1940-6029 |
Keywords
- Bioconjugation
- Chaperone protein Hsp90
- Protein dynamics
- Protein purification
- Single-molecule FRET
- Surface functionalization
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Single-Molecule FRET-Resolved Protein Dynamics - from Plasmid to Data in Six Steps
Vermeer, B. (Creator), van Ossenbruggen, J. (Creator) & Schmid, S. (Creator), Wageningen University & Research, 3 Jul 2023
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