Updated publication reference for PubMed record(s): 31953415. Updated publication reference for PubMed record(s): 31953415. The ATP-dependent chaperones of the Hsp70 class (DnaK in E. coli) function in protein folding in cooperation with J proteins and nucleotide exchange factors (DnaJ and GrpE in E. coli, respectively). Hsp70 prevents protein aggregation, increasing the folding yield, but whether it also enhances the rate of folding is unclear. Equilibrium hydrogen/deuterium exchange – mass spectrometry showed that DnaK stabilizes a poorly structured state of firefly luciferase (FLuc). Pulsed-label experiments, together with orthogonal analyses by spFRET, identified compact inter-domain misfolded states of FLuc that convert slowly to the native state. DnaK binding expands the misfolded region and thereby resolves the kinetically-trapped intermediates, with folding occurring upon GrpE-mediated release. By resolving misfolding and accelerating folding the Hsp70 system can maintain proteins in their native states under otherwise denaturing stress conditions.