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 / 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 -helical proteins forming a number of H-bonds
proportional to the amount of surface buried in the transition state.
/
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 
