Thermodynamic characterization and folding of a moderately thermophilic RNase H: Identification of factors that lead to adaptation at different temperatures

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 T. thermophilus and the mesophile E. coli revealed that the thermostability arises in part from an unusually low change in heat capacity upon unfolding (DeltaCp) for the thermophilic protein [Hollien J. and Marqusee S. (1999) Biochemistry 38, 3831-3836]. I have further examined how nearly identical proteins can adapt to different thermal constraints by adding a moderately thermophilic homolog to the previously characterized mesophilic and thermophilic pair. I identified a putative RNase H from C. tepidum and demonstrated that it is an active RNase H and adopts the RNase H fold. The moderately thermophilic protein has a melting temperature (Tm) similar to that of the mesophilic homolog, yet also has a surprisingly low DeltaCp, like the thermophilic homolog. I have also shown that the new RNase H folds through a pathway similar to that of the previously studied RNases H, populating a folding intermediate. I have taken the folding core of C. tepidum RNase H and through the creation of chimeric proteins determined that it is indeed the region that dictates the DeltaCp of the protein. Interestingly, the increased stability of the folding core led to the creation of a new RNase H with increased stability from either parent protein and a more thermophilic stability profile. These results suggest that lowering the DeltaCp may be a general strategy for achieving thermophilicity for some protein families and that the folding core is 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 DeltaCp, and therefore fine-tune the energy landscape in a predictable fashion.