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

摘要

Proteins from thermophilic organisms are able to function under conditions that render a typicalnmesophilic protein inactive. Pairwise comparisons of homologous mesophilic and thermophilic proteins cannhelp to identify the energetic features of a protein’s energy landscape that lead to such thermostability.nPrevious studies of bacterial ribonucleases H (RNases H) from the thermophile Thermus thermophilusnand the mesophile Escherichia coli revealed that the thermostability arises in part from an unusually lownchange in heat capacity upon unfolding (ΔCp) for the thermophilic protein [Hollien, J., and Marqusee, S.n(1999) Biochemistry 38, 3831-3836]. Here, we have further examined how nearly identical proteins cannadapt to different thermal constraints by adding a moderately thermophilic homologue to the previouslyncharacterized mesophilic and thermophilic pair.We identified a putative RNase H from Chlorobium. tepidumnand demonstrated that it is an active RNase H and adopts the RNase H fold. The moderately thermophilicnprotein has amelting temperature (Tm) similar to that of themesophilic homologue yet also has a surprisinglynlow ΔCp, like the thermophilic homologue. This new RNase H folds through a pathway similar to that ofnthe previously studied RNases H. These results suggest that lowering the ΔCp may be a generalnstrategy for achieving thermophilicity for some protein families and implicate the folding core as the majorncontributor to this effect. It should now be possible to design RNases H that display the desired thermophilicnor mesophilic properties, as defined by their ΔCp values, and therefore fine-tune the energy landscapenin a predictable fashion