biostudies-literatureUnknown11(1)Imamoglu RThe 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 Nature communications365https://www.ebi.ac.uk/biostudies/studies/S-EPMC6969021biostudies-literatureBacterial Hsp70 resolves misfolded states and accelerates productive folding of a multi-domain protein.PMC6969021Hayer-Hartl MHartl FUImamoglu RBalchin DfalseBacterial 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 2020-01-01T00:00:00Z2020 Jan2026-04-29T16:07:59.946Z2020-05-22T08:31:41ZS-EPMC69690213195341510.1038/s41467-019-14245-4