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

Lena Voith von Voithenberg, Anders Barth, Vanessa Trauschke, Benjamin Demarco, Swati Tyagi, Christine Koehler, Edward A. Lemke, Don C. Lamb*

*Corresponding author for this work

Research output: Contribution to journalArticleScientificpeer-review

7 Citations (Scopus)


Cellular function depends on the correct folding of proteins inside the cell. Heat-shock proteins 70 (Hsp70s), being among the first molecular chaperones binding to nascently translated proteins, aid in protein folding and transport. They undergo large, coordinated intra- and interdomain structural rearrangements mediated by allosteric interactions. Here, we applied a three-color single-molecule Förster resonance energy transfer (FRET) combined with three-color photon distribution analysis to compare the conformational cycle of the Hsp70 chaperones DnaK, Ssc1, and BiP. By capturing three distances simultaneously, we can identify coordinated structural changes during the functional cycle. Besides the known conformations of the Hsp70s with docked domains and open lid and undocked domains with closed lid, we observed additional intermediate conformations and distance broadening, suggesting flexibility of the Hsp70s in adopting the states in a coordinated fashion. Interestingly, the difference of this distance broadening varied between DnaK, Ssc1, and BiP. Study of their conformational cycle in the presence of substrate peptide and nucleotide exchange factors strengthened the observation of additional conformational intermediates, with BiP showing coordinated changes more clearly compared to DnaK and Ssc1. Additionally, DnaK and BiP were found to differ in their selectivity for nucleotide analogs, suggesting variability in the recognition mechanism of their nucleotide-binding domains for the different nucleotides. By using three-color FRET, we overcome the limitations of the usual single-distance approach in single-molecule FRET, allowing us to characterize the conformational space of proteins in higher detail.

Original languageEnglish
Article numbere2025578118
Number of pages12
JournalProceedings of the National Academy of Sciences of the United States of America
Issue number33
Publication statusPublished - 2021
Externally publishedYes


  • Conformational dynamics
  • Coordinated motion
  • Heat-shock proteins
  • Single-molecule Förster resonance energy transfer
  • Three-color photon distribution analysis

Cite this