Confirmation of the hierarchical folding of RNase H: a protein engineeringstudy

The kinetic intermediate of RNase H is structured in a core region of the p rotein. To probe the role of this intermediate in the folding of RNase ii, the folding kinetics of mutant proteins with altered native state stabiliti es were investigated. Mutations within the folding core destabilize the kin etic intermediate and slow refolding in a manner consistent with an obligat ory intermediate model. Mutations outside of the folding core, however, do not affect the stability of the kinetic intermediate but do perturb the nat ive state and transition state. These results indicate that interactions fo rmed in the intermediate persist in the transition and native states and th at RNase H folds through a hierarchical mechanism.