Identification of residual structure in the unfolded state of ribonuclease H1 from the moderately thermophilic Chlorobium tepidum: comparison with thermophilic and mesophilic homologues.

Ribonucleases H from organisms that grow at different temperatures demonstrate a variable change in heat capacity upon unfolding (DeltaC degrees (P)) [Ratcliff, K., et al. (2009) Biochemistry 48, 5890-5898]. This DeltaC degrees (P) has been shown to correlate with a tolerance to higher temperatures and residual structure in the unfolded state of the thermophilic proteins. In the RNase H from Thermus thermophilus, the low DeltaC degrees (P) has been shown to arise from the same region as the folding core of the protein, and mutagenic studies have shown that loss of a hydrophobic residue in this region can disrupt this residual unfolded state structure and result in a return to a more mesophile-like DeltaC degrees (P) [Robic, S., et al. (2002) Protein Sci. 11, 381-389; Robic, S., et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 11345-11349]. To understand further how residual structure in the unfolded state is encoded in the sequences of these thermophilic proteins, we subjected the RNase H from Chlorobium tepidum to similar studies. Analysis of new chimeric proteins reveals that like T. thermophilus RNase H, the folding core of C. tepidum RNase H plays an important role in the unfolded state of this protein. Mutagenesis studies, based on both a computational investigation of the hydrophobic networks in the core region and comparisons with similar studies on T. thermophilus RNase H, identify new residues involved in this residual structure and suggest that the residual structure in the unfolded state of C. tepidum RNase H is more restricted than that of T. thermophilus. We conclude that while the folding core region determines the thermophilic-like behavior of this family of proteins, the residue-specific details vary.