Stopped-flow fluorescence studies on the N-terminal domain of rat CD2 (CD2.d1) have demonstrated that folding from the fully denatured state (U) proceeds via the transient accumulation of an apparent intermediate (I) in a so-called burst phase that precedes the rate-limiting transition leading to the native state (N). A previous pH-dependent equilibrium hydrogen exchange (HX) study identified a subset of amides in CD2.d1 which, under EX2 conditions, exchange from N with free energies greater than or equal to the free energy 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 an asymptote that matches the global unfolding rate calculated from the stopped-flow data, suggesting that exchange for these amides requires traversing the N-to-I transition state barrier. Exchange for these amides presumably occurs from exchange-competent forms comprising the kinetic burst phase therefore. 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 data fit to a two-component, sub-global (sg)/global (g) NHX mechanism, yielding Delta G and m value parameters (where the m value is a measure of hydrocarbon solvation). Regression analysis demonstrates that the (m(sg), Delta G(sg)) and (m(g), Delta G(g)) values calculated for this subset of amides correspond with those describing the kinetic burst phase transition. This result confirms the ability of the NHX technique to explore the structural and energetic properties of kinetic folding intermediates.
Copyright 2001 Academic Press.