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? 2000 Nature America Inc. ? insight D/H amide kinetic isotope effects reveal when hydrogen bonds form during protein folding Bryan A. Krantz1, Liam B. Moran1,2, Alex Kentsis1,3 and Tobin R. Sosnick1 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 ~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 ~50% of the native H-bonds are formed in the transition state of these helical proteins. In contrast, an α/β 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 ~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 α-helical proteins forming a number of H-bonds proportional to the amount of surface buried in the transition state. .com The H-bond network is one of the defining aspects of a protein’s favorable hydrophobic burial14. Because of the variety of features structure. Although the net effect on protein stability is unclear, affected, it is difficult to unambiguously extract information nearly all buried carbonyls and amides are required to form H- regarding the degree of H-bond formation in the TS using this bonds, thereby restricting the architecture of allowed protein cosolvent. folds. At what point backbone H-bonds form during a two