title: A single mutation at residue 25 populates the folding intermediate of E. coli RNase H and reveals a highly dynamic partially folded ensemble.
abstract: Understanding the nature of partially folded intermediates transiently populated during protein folding is important for understanding both protein folding and misfolding. These ephemeral species, however, often elude direct experimental characterization. The well-characterized protein ribonuclease H (RNase H) from Escherichia coli populates an on-pathway intermediate identified in both bulk studies and single-molecule mechanical unfolding experiments. Here, we set out to trap the transient intermediate of RNase H at equilibrium by selectively destabilizing the region of the protein known to be unfolded in this species. Surprisingly, a single change at Ile25 (I25A) resulted in the equilibrium population of the intermediate under near-native conditions. The intermediate was undetectable in a series of heteronuclear single quantum coherences, revealing the dynamic nature of this partially unfolded form on the timescale of NMR detection. This result is in contrast to studies in which the structures of trapped intermediates are solved by NMR, indicating that they are well packed and native-like. The dynamic nature of the RNase H intermediate may be important for its role as an on-pathway, productive species that promotes efficient folding.
authors.affiliation: Institute for Quantitative Biosciences-Berkeley, University of California, Berkeley, CA 94720-3220, USA.
authors.forename: Katelyn B
authors.initials: KB
authors.lastname: Connell
authors.forename: Geoffrey A
authors.initials: GA
authors.lastname: Horner
authors.forename: Susan
authors.initials: S
authors.lastname: Marqusee
mesh_terms: D019943:Amino Acid Substitution; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology*; D007532:Isoleucine / Q000737:chemistry* / Q000235:genetics; D008956:Models, Chemical; D009154:Mutation; D011487:Protein Conformation; D015202:Protein Engineering; D017510:Protein Folding; D016914:Ribonuclease H / Q000737:chemistry* / Q000235:genetics
chemical_list: D007532:Isoleucine; D016914:Ribonuclease H; C078856:ribonuclease HI