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 seco ndary and tertiary structural features in a so-called burst phase, precedin g the relatively slow, highly activated transition leading to the native st ate. A fundamental question is: do these burst phase reactions comprise two phase-separated thermodynamic states or a continuum of states? Ribonucleas e HI (RNase H) from Escherichia coli and phage T4 lysozyme (T4L) both exhib it such a phenomenon. Native-state hydrogen exchange (NHX) data have been c ollected 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 stat e (Delta G(sg) and m(sg), where the sg subscript denotes sub-global). There is good evidence that these parameters pertain to exchange-competent speci es comprising the burst phase observed in the global folding kinetics. We c ombine the results from the global folding kinetics of these proteins with a statistical analysis of their NHX parameters to determine if the distribu tion of experimental (m(sg), Delta G(sg)) values derive from a mechanism wh ere the burst phase is two-state. For RNase H, this analysis demonstrates t hat the burst phase of this protein is not two-state; the results imply a d istribution of states, m and Delta G exhibiting a linear functional relatio nship consistent with the global folding parameters. For T4L, it is difficu lt to distinguish the observed distribution of m(sg), Delta G(sg) values fr om that expected for a mechanism where the burst phase is two-state. The re sults for RNase H* lend support for the idea that the burst phase reaction of this protein comprises a continuum of states. This has important implica tions for how we model the process of structural acquisition in protein fol ding reactions. (C) 2000 Academic Press.