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Protein folding is an indispensable process for the majority of proteinsafter their synthesis from ribosomes in the cell. Most in vitro protein foldingstudies have focused on single-domain proteins. Hence, it is important tounderstand the folding process of multi-domain proteins, especially whendomains are discontinuous. We choose the Maltose binding protein (MBP) as amodel system. In particular, we studied a mutant of MBP that folds slowly.Here, using two- and three-color single-molecule Foerster resonance energytransfer (smFRET) experiments, we study the refolding of both the domains andthe interaction between the domains of DM-MBP. Initial two-color smFRETmeasurements of the N-terminal domain (NTD) reveal the presence of a foldingintermediate. The same folding intermediate is observed in measurementsmonitoring the C- terminal domain (CTD) and the NTD-CTD (N-C) interface. Therefolding intermediate is dynamic on the sub-millisecond timescale.Quantitative analysis on underlying dynamic interconversions revealed a delayin NTD folding imposed by the entropic barrier being the primary cause for slowDM-MBP folding. Moreover, CTD folds after NTD completes the folding. Usingthree-color smFRET, we could show the NTD folds first and CTD later in a sameprotein. Molecular dynamic simulations for temperature-induced unfolding on WT-and DM-MBP identify a folding nucleus in the NTD, which is rich in hydrophobicresidues, and explains why the two mutations slow down the folding kinetics. Inthe presence of the bacterial Hsp60 chaperonin system GroEL/ES, we observe thatDM-MBP is dynamic within the chaperonin cavity but the chaperonin limits theconformational space of substrate. Hence, confinement aids DM-MBP in overcomingthe entropic barrier. The study reports on the subtle tuning and co-dependencyfor protein folding between two-domains with a discontinuous arrangement.

A dynamic intermediate state limits the folding rate of a discontinuous two-domain protein