Edited by George H. Lorimer, University of Maryland, College Park, MD, and approved December 16, 2015 (received for review September 29, 2015)
Understanding protein folding is, as yet, an unsolved question in the life sciences that has relevance for many diseases. While the folding of simple and small protein domains is well studied, for large proteins, the abundance of pathways and intermediate states makes them difficult to characterize using standard protein folding experiments. With single-molecule optical tweezers experiments, we can overcome these limitations. We observe in real time the folding of a dimeric, three-domain protein from the fully unfolded chain to the biologically active, quaternary structure. The likelihood of the folding process being hindered by misfolded intermediates increases with chain length. These misfolded states can slow down folding significantly and may lead to aggregation in vivo.
Folding of small proteins often occurs in a two-state manner and is well understood both experimentally and theoretically. However, many proteins are much larger and often populate misfolded states, complicating their folding process significantly. Here we study the complete folding and assembly process of the 1,418 amino acid, dimeric chaperone Hsp90 using single-molecule optical tweezers. Although the isolated C-terminal domain shows two-state folding, we find that the isolated N-terminal as well as the middle domain populate ensembles of fast-forming, misfolded states. These intradomain misfolds slow down folding by an order of magnitude. Modeling folding as a competition between productive and misfolding pathways allows us to fully describe the folding kinetics. Beyond intradomain misfolding, folding of the full-length protein is further slowed by the formation of interdomain misfolds, suggesting that with growing chain lengths, such misfolds will dominate folding kinetics. Interestingly, we find that small stretching forces applied to the chain can accelerate folding by preventing the formation of cross-domain misfolding intermediates by leading the protein along productive pathways to the native state. The same effect is achieved by cotranslational folding at the ribosome in vivo.
Author contributions: M.J., J.B., T.H., and M.R. designed research; M.J. performed research; M.J. and M.R. analyzed data; and M.J., J.B., T.H., and M.R. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1518827113/-/DCSupplemental.
