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

Biophysical Journal
Zackary N SchollPiotr E Marszalek

Abstract

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 sequence...Continue Reading

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Citations

Nov 30, 2019·Proceedings of the National Academy of Sciences of the United States of America·Kaixian LiuChristian M Kaiser
Dec 31, 2020·International Journal of Molecular Sciences·Yue DingPiotr Marszalek

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