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Proc Natl Acad Sci U S A. 2016 Apr 26;113(17):4747-52. doi: 10.1073/pnas.1522500113. Epub 2016 Apr 13.

Cooperative folding near the downhill limit determined with amino acid resolution by hydrogen exchange.

Author information

1
Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL 60637;
2
Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL 60637; Institute for Biophysical Dynamics, University of Chicago, Chicago, IL 60637;
3
Department of Chemistry, University of Chicago, Chicago, IL 60637; James Franck Institute, University of Chicago, Chicago, IL 60637; Computation Institute, University of Chicago, Chicago, IL 60637 trsosnic@uchicago.edu freed@uchicago.edu.
4
Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL 60637; Institute for Biophysical Dynamics, University of Chicago, Chicago, IL 60637; Computation Institute, University of Chicago, Chicago, IL 60637 trsosnic@uchicago.edu freed@uchicago.edu.

Abstract

The relationship between folding cooperativity and downhill, or barrier-free, folding of proteins under highly stabilizing conditions remains an unresolved topic, especially for proteins such as λ-repressor that fold on the microsecond timescale. Under aqueous conditions where downhill folding is most likely to occur, we measure the stability of multiple H bonds, using hydrogen exchange (HX) in a λYA variant that is suggested to be an incipient downhill folder having an extrapolated folding rate constant of 2 × 10(5) s(-1) and a stability of 7.4 kcal·mol(-1) at 298 K. At least one H bond on each of the three largest helices (α1, α3, and α4) breaks during a common unfolding event that reflects global denaturation. The use of HX enables us to both examine folding under highly stabilizing, native-like conditions and probe the pretransition state region for stable species without the need to initiate the folding reaction. The equivalence of the stability determined at zero and high denaturant indicates that any residual denatured state structure minimally affects the stability even under native conditions. Using our ψ analysis method along with mutational ϕ analysis, we find that the three aforementioned helices are all present in the folding transition state. Hence, the free energy surface has a sufficiently high barrier separating the denatured and native states that folding appears cooperative even under extremely stable and fast folding conditions.

KEYWORDS:

cooperative folding; folding pathway; hydrogen exchange; protein folding; λ repressor

PMID:
27078098
PMCID:
PMC4855600
DOI:
10.1073/pnas.1522500113
[Indexed for MEDLINE]
Free PMC Article

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