Single-Molecule FRET-Resolved Protein Dynamics – from Plasmid to Data in Six Steps

Benjamin Vermeer, Jannick van Ossenbruggen, Sonja Schmid*

*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceedingChapterAcademicpeer-review


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 languageEnglish
Title of host publicationMethods in Molecular Biology
PublisherHumana Press
Number of pages25
ISBN (Electronic)9781071633779
ISBN (Print)9781071633762
Publication statusPublished - 2024

Publication series

NameMethods in Molecular Biology
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029


  • Bioconjugation
  • Chaperone protein Hsp90
  • Protein dynamics
  • Protein purification
  • Single-molecule FRET
  • Surface functionalization


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