D/H amide kinetic isotope effects reveal when hydrogen bonds form during protein folding

We have exploited a procedure to identify when hydrogen bonds (H-bonds) for m under two-state folding conditions using equilibrium and kinetic deuteriu m/hydrogen amide isotope effects. Deuteration decreases the stability of eq uine cytochrome c and the dimeric and crosslinked versions of the GCN4-p1 c oiled coil by similar to 0.5 kcal mol(-1). For all three systems, the decre ase in equilibrium stability is reflected by a decrease in refolding rates and a near equivalent increase in unfolding rates. This apportionment indic ates that similar to 50% of the native H-bonds are formed in the transition state of these helical proteins. In contrast, an alpha/beta protein, mamma lian ubiquitin, exhibits a small isotope effect only on unfolding rates, su ggesting its folding pathway may be different. These four proteins recapitu late the general trend that similar to 50% of the surface buried in the nat ive state is buried in the transition state, leading to the hypothesis that H-bond formation in the transition state is cooperative, with alpha-helica l proteins forming a number of H-bonds proportional to the amount of surfac e buried in the transition state.