A kinetic folding intermediate probed by native state hydrogen exchange

Stopped-flow fluorescence studies on the N-terminal domain of rat CD2 (CD2. d1) have demonstrated that folding from the fully denatured state (U) proce eds via the transient accumulation of an apparent intermediate (I) in a so- called burst phase that precedes the rate-limiting transition leading to th e native state (N). A previous pH-dependent equilibrium hydrogen exchange ( HX) study identified a subset of amides in CD2.d1 which, under EX2 conditio ns, exchange from N with free energies greater than or equal to the free en ergy difference between the N and I states calculated from the stopped-flow data. Under EX1 conditions the rates of HX for these amides tend towards a n asymptote that matches the global unfolding rate calculated from the stop ped-flow data, suggesting that exchange for these amides requires traversin g the N-to-I transition state barrier. Exchange for these amides presumably occurs from exchange-competent forms comprising the kinetic burst phase th erefore. To explore this idea further, native state HX (NHX) data have been collected for CD2.d1 under EX2 conditions using denaturant concentrations which span either side of the denaturant concentration where, according to the stopped-flow data, the apparent U and I states are iso-energetic. The d ata to a two-component, sub-global (sg)/global (g) NHX mechanism, yielding DeltaG and m value parameters (where the rn value is a measure of hydrocarb on solvation). Regression analysis demonstrates that the (m(g), DeltaG(sg)) and (m(g), DeltaG(g)) values calculated for this subset of amides correspo nd with those describing the kinetic burst phase transition. This result co nfirms the ability of the NHX technique to explore the structural and energ etic properties of kinetic folding intermediates. (C) 2001 Academic Press.