Comparative analysis of the coordinated motion of Hsp70s from different organelles observed by single-molecule three-color FRET

Significance The coordinated motion and conformational changes are fundamental for the function of many macromolecules. However, obtaining direct evidence of coordinated changes between different subdomains within an individual molecule remains challenging. Here, we apply a single-molecule three-color Förster resonance energy transfer to simultaneously measure three distances within a single molecule. We utilized this method to analyze the coordinated motion of heat-shock proteins (Hsps). Excitingly, our study provides direct evidence for key differences in the conformational cycle of different Hsp70s despite their high evolutionary conservation. This work paves the way to relate coordinated motion and allosteric effects to molecular function.