Equilibrium Population of the Folding Intermediate of RNase H and its Importance in the Folding Trajectory

Abstract

Proteins can sample a variety of partially folded conformations during the transition between the unfolded and native states. Some proteins never significantly populate these high-energy states and fold by an apparently two-state process. What factors govern which conformations are accessible to a protein as it folds? We have attempted to re-route the folding of ribonuclease H from E. coli by manipulating its regional stability. Using phi-value analysis, we compare the structures of the transition states for folding of RNases H that fold with and without a detectable partially folded intermediate and find that both versions of RNase H fold through a similar trajectory with similar high energy conformations. In light of the general importance of this species on the folding pathway, we attempted to populate the intermediate at equilibrium by destabilizing the region of the protein that is unfolded in this form. Surprisingly, a single change at Ile 25 (I25A) resulted in the equilibrium population of the intermediate under near-native conditions. The intermediate was undetectable in a series of HSQC's, revealing the dynamic nature of this partially unfolded form on the timescale of NMR detection. The dynamic nature of the RNase H intermediate may be important for its role as an on-pathway, productive species that promotes efficient folding.