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

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.