Single-Molecule Dynamics of an HSP70 Chaperone

Abstract

Hsp70s are highly dynamic nanomachines that modulate the conformations of their substrate polypeptides by transiently binding to short hydrophobic stretches. In order to investigate the conformational dynamics of Hsp70 during its chaperone cycle and the transient nature of Hsp70-substrate interaction, we fused a fragment of sigma-32 protein via a 30-residue long linker to the C-terminus of DnaK. We employ dual-beam optical tweezers setup to observe the kinetics of the tethered peptide in the presence of nucleotides and co-chaperones to understand the DnaK/DanJ/GrpE foldase machinery. Under constant mechanical load, the rapid association and dissociation of the tethered peptide and the peptide-induced ATP-hydrolysed conformation of DnaK were observed. Our concentration-dependent experiments of GrpE also show that GrpE binding to the complex stimulates the exchange of ADP to ATP and DnaK remains in its low-affinity conformation at high concentration of GrpE. Mutant (T199A) analysis furthermore revealed that DnaJ can also act as a substrate and induce ATP-hydrolysis at high concentrations. In conclusion, our findings provide a better understanding of the Hsp70 chaperone cycle and its regulation in the presence of substrates and co-chaperone.