Interactions in the folding intermediate of Escherichia coli RNase H: Comparisons with the native state ensemble

Some kinetic intermediates appear to be subsets of the native state, supporting a hierarchical model of folding. How similar are these intermediates to the native state ensemble? This dissertation focuses on specific interactions in the early folding intermediate of E. coli RNase H, and how they compare to the native state. I address the role of electrostatic interactions in the kinetic intermediate by mutating a partially buried, salt bridge network. In this variant, the native state is selectively destabilized, causing the kinetic intermediate, usually only transiently populated, to be present at equilibrium. The sole indicator of this deviation from two-state behavior was the change in the denaturant dependence of the free energy of unfolding, or “m value”. These results suggest electrostatic interactions may not be formed in the kinetic intermediate. I also compare interactions at the packing interface of two core helices (A and D) in both the kinetic intermediate and the native state core by characterizing a series of hydrophobic mutations at a residue at this interface, Ile 53. Comparisons to the native state using “&phgr; value analysis” demonstrate that although the kinetic intermediate is more loosely-packed than the native state, steric changes can be destabilizing, supporting a role of packing interactions in the intermediate. Characterization of one of these core mutations (Ile 53 to Ala) by native state hydrogen exchange strengthens the resemblance of the kinetic intermediate to a partially unfolded form (PUF) in the native state ensemble, as both forms are destabilized in the variant. This supports a hierarchical model of folding, as the PUF is a subset of the native state. These experiments show a localized destabilization of the core upon the point mutation, however, an increase is seen in the denaturant-independent, “local fluctuations” in the periphery. The studies in this dissertation characterize specific tertiary interactions in the kinetic intermediate, and further tighten the link to the PUF found in the native state ensemble. This work supports a hierarchical model of folding, in which early interactions in the kinetic intermediate are built upon in the high-energy transition state in order to achieve the native state.