We have exploited a procedure to identify when hydrogen bonds (H-bonds) form under two-state folding conditions using equilibrium and kinetic deuterium/hydrogen amide isotope effects. Deuteration decreases the stability of equine cytochrome c and the dimeric and crosslinked versions of the GCN4-p1 coiled coil by approximately 0. 5 kcal mol-1. For all three systems, the decrease in equilibrium stability is reflected by a decrease in refolding rates and a near equivalent increase in unfolding rates. This apportionment indicates that approximately 50% of the native H-bonds are formed in the transition state of these helical proteins. In contrast, an alpha/beta protein, mammalian ubiquitin, exhibits a small isotope effect only on unfolding rates, suggesting its folding pathway may be different. These four proteins recapitulate the general trend that approximately 50% of the surface buried in the native state is buried in the transition state, leading to the hypothesis that H-bond formation in the transition state is cooperative, with alpha-helical proteins forming a number of H-bonds proportional to the amount of surface buried in the transition state.

• PMID: 10625430
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Journal Article | Research Support, Non-U.S. Gov't | Research Support, U.S. Gov't, P.H.S.

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Krantz BA, Moran LB, Kentsis A, Sosnick TR

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