A statistical appraisal of native state hydrogen exchange data: evidence for a burst phase continuum?

For a number of proteins, folding occurs via the rapid accumulation of secondary and tertiary structural features in a so-called burst phase, preceding the relatively slow, highly activated transition leading to the native state. A fundamental question is: do these burst phase reactions comprise two phase-separated thermodynamic states or a continuum of states? Ribonuclease HI (RNase H) from Escherichia coli and phage T4 lysozyme (T4L) both exhibit such a phenomenon. Native-state hydrogen exchange (NHX) data have been collected for these proteins, providing residue-specific free energies and m-values (a measure of hydrocarbon solvation) for the manifold of partially unfolded, exchange-competent forms that are accessible from the native state (DeltaG(sg) and m(sg), where the sg subscript denotes sub-global). There is good evidence that these parameters pertain to exchange-competent species comprising the burst phase observed in the global folding kinetics. We combine the results from the global folding kinetics of these proteins with a statistical analysis of their NHX parameters to determine if the distribution of experimental (m(sg), DeltaG(sg)) values derive from a mechanism where the burst phase is two-state. For RNase H, this analysis demonstrates that the burst phase of this protein is not two-state; the results imply a distribution of states, m and DeltaG exhibiting a linear functional relationship consistent with the global folding parameters. For T4L, it is difficult to distinguish the observed distribution of m(sg), DeltaG(sg) values from that expected for a mechanism where the burst phase is two-state. The results for RNase H* lend support for the idea that the burst phase reaction of this protein comprises a continuum of states. This has important implications for how we model the process of structural acquisition in protein folding reactions.