It is estimated that two-thirds of all proteins in higher organisms are composed of multiple domains, many of them containing discontinuous folds. However, to date, most in vitro protein folding studies have focused on small, single-domain proteins. As a model system for a two-domain discontinuous protein, we study the unfolding/refolding of a slow-folding double mutant of the maltose binding protein (DM-MBP) using single-molecule two- and three-color Förster Resonance Energy Transfer experiments. We observe a dynamic folding intermediate population in the N-terminal domain (NTD), C-terminal domain (CTD), and at the domain interface. The dynamic intermediate fluctuates rapidly between unfolded states and compact states, which have a similar FRET efficiency to the folded conformation. Our data reveals that the delayed folding of the NTD in DM-MBP is imposed by an entropic barrier with subsequent folding of the highly dynamic CTD. Notably, accelerated DM-MBP folding is routed through the same dynamic intermediate within the cavity of the GroEL/ES chaperone system, suggesting that the chaperonin limits the conformational space to overcome the entropic folding barrier. Our study highlights the subtle tuning and co-dependency in the folding of a discontinuous multi-domain protein.

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.