Bacterial Hsp70 resolves misfolded states and accelerates productive folding of a multi-domain protein

The ATP-dependent Hsp70 chaperones (DnaK in E. coli) mediate protein folding in cooperation with J proteins and nucleotide exchange factors (E. coli DnaJ and GrpE, respectively). The Hsp70 system prevents protein aggregation and increases folding yields. Whether it also enhances the rate of folding remains unclear. Here we show that DnaK/DnaJ/GrpE accelerate the folding of the multi-domain protein firefly luciferase (FLuc) 20-fold over the rate of spontaneous folding measured in the absence of aggregation. Analysis by single-pair FRET and hydrogen/deuterium exchange identified inter-domain misfolding as the cause of slow folding. DnaK binding expands the misfolded region and thereby resolves the kinetically-trapped intermediates, with folding occurring upon GrpE-mediated release. In each round of release DnaK commits a fraction of FLuc to fast folding, circumventing misfolding. We suggest that by resolving misfolding and accelerating productive folding, the bacterial Hsp70 system can maintain proteins in their native states under otherwise denaturing stress conditions. The Hsp70 system prevents protein aggregation and increases folding yields, but it is unknown whether it also enhances the rate of folding. Here the authors combine refolding assays, FRET and hydrogen/deuterium exchange-mass spectrometry measurements to study the folding of firefly luciferase and find that the bacterial Hsp70 actively promotes the folding of this multi-domain protein.