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J Am Chem Soc. 2014 Jan 15;136(2):733-40. doi: 10.1021/ja410437d. Epub 2013 Dec 31.

Ultrafast hydrogen exchange reveals specific structural events during the initial stages of folding of cytochrome c.

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1
Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, United States.

Abstract

Many proteins undergo a sharp decrease in chain dimensions during early stages of folding, prior to the rate-limiting step in folding. However, it remains unclear whether compact states are the result of specific folding events or a general hydrophobic collapse of the poly peptide chain driven by the change in solvent conditions. To address this fundamental question, we extended the temporal resolution of NMR-detected H/D exchange labeling experiments into the microsecond regime by adopting a microfluidics approach. By observing the competition between H/D exchange and folding as a function of labeling pH, coupled with direct measurement of exchange rates in the unfolded state, we were able to monitor hydrogen-bond formation for over 50 individual backbone NH groups within the initial 140 microseconds of folding of horse cytochrome c. Clusters of solvent-shielded amide protons were observed in two α-helical segments in the C-terminal half of the protein, while the N-terminal helix remained largely unstructured, suggesting that proximity in the primary structure is a major factor in promoting helix formation and association at early stages of folding, while the entropically more costly long-range contacts between the N- and C-terminal helices are established only during later stages. Our findings clearly indicate that the initial chain condensation in cytochrome c is driven by specific interactions among a subset of α-helical segments rather than a general hydrophobic collapse.

PMID:
24364692
PMCID:
PMC3956590
DOI:
10.1021/ja410437d
[Indexed for MEDLINE]
Free PMC Article
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