Competing Pathways and Multiple Folding Nuclei in a Large Multidomain Protein, Luciferase.

Journal Article (Journal Article)

Proteins obtain their final functional configuration through incremental folding with many intermediate steps in the folding pathway. If known, these intermediate steps could be valuable new targets for designing therapeutics and the sequence of events could elucidate the mechanism of refolding. However, determining these intermediate steps is hardly an easy feat, and has been elusive for most proteins, especially large, multidomain proteins. Here, we effectively map part of the folding pathway for the model large multidomain protein, Luciferase, by combining single-molecule force-spectroscopy experiments and coarse-grained simulation. Single-molecule refolding experiments reveal the initial nucleation of folding while simulations corroborate these stable core structures of Luciferase, and indicate the relative propensities for each to propagate to the final folded native state. Both experimental refolding and Monte Carlo simulations of Markov state models generated from simulation reveal that Luciferase most often folds along a pathway originating from the nucleation of the N-terminal domain, and that this pathway is the least likely to form nonnative structures. We then engineer truncated variants of Luciferase whose sequences corresponded to the putative structure from simulation and we use atomic force spectroscopy to determine their unfolding and stability. These experimental results corroborate the structures predicted from the folding simulation and strongly suggest that they are intermediates along the folding pathway. Taken together, our results suggest that initial Luciferase refolding occurs along a vectorial pathway and also suggest a mechanism that chaperones may exploit to prevent misfolding.

Full Text

Duke Authors

Cited Authors

  • Scholl, ZN; Yang, W; Marszalek, PE

Published Date

  • May 2017

Published In

Volume / Issue

  • 112 / 9

Start / End Page

  • 1829 - 1840

PubMed ID

  • 28494954

Pubmed Central ID

  • PMC5425382

Electronic International Standard Serial Number (EISSN)

  • 1542-0086

International Standard Serial Number (ISSN)

  • 0006-3495

Digital Object Identifier (DOI)

  • 10.1016/j.bpj.2017.03.028


  • eng