Structure, Stability, and Folding of Ribonuclease H1 from the Moderately Thermophilic Chlorobium tepidum: Comparison with Thermophilic and Mesophilic Homologues

Abstract

Proteins from thermophilic organisms are able to function under conditions that render a typical mesophilic protein inactive. Pairwise comparisons of homologous mesophilic and thermophilic proteins can help to identify the energetic features of a protein’s energy landscape that lead to such thermostability. Previous studies of bacterial ribonucleases H (RNases H) from the thermophile Thermus thermophilus and the mesophile Escherichia coli revealed that the thermostability arises in part from an unusually low change in heat capacity upon unfolding (ΔC_p) for the thermophilic protein [Hollien, J., and Marqusee, S. (1999) Biochemistry 38, 3831−3836]. Here, we have further examined how nearly identical proteins can adapt to different thermal constraints by adding a moderately thermophilic homologue to the previously characterized mesophilic and thermophilic pair. We identified a putative RNase H from Chlorobium. tepidum and demonstrated that it is an active RNase H and adopts the RNase H fold. The moderately thermophilic protein has a melting temperature (T_m) similar to that of the mesophilic homologue yet also has a surprisingly low ΔC_p, like the thermophilic homologue. This new RNase H folds through a pathway similar to that of the previously studied RNases H. These results suggest that lowering the ΔC_p may be a general strategy for achieving thermophilicity for some protein families and implicate the folding core as the major contributor to this effect. It should now be possible to design RNases H that display the desired thermophilic or mesophilic properties, as defined by their ΔC_p values, and therefore fine-tune the energy landscape in a predictable fashion.