Contributions of folding cores to the thermostabilities of two ribonucleases H.
To investigate the contribution of the folding cores to the thermodynamic stability of RNases H, we used rational design to create two chimeras composed of parts of a thermophilic and a mesophilic RNase H. Each chimera combines the folding core from one parent protein and the remaining parts of the other. Both chimeras form active, well-folded RNases H. Stability curves, based on CD-monitored chemical denaturations, show that the chimera with the thermophilic core is more stable, has a higher midpoint of thermal denaturation, and a lower change in heat capacity (DeltaCp) upon unfolding than the chimera with the mesophilic core. A possible explanation for the low DeltaCp of both the parent thermophilic RNase H and the chimera with the thermophilic core is the residual structure in the denatured state. On the basis of the studied parameters, the chimera with the thermophilic core resembles a true thermophilic protein. Our results suggest that the folding core plays an essential role in conferring thermodynamic parameters to RNases H.
Robic
Srebrenka
S
Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA.
Berger
James M
JM
Marqusee
Susan
S
D000595:Amino Acid Sequence; D002678:Chimera; D002942:Circular Dichroism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology; D006358:Hot Temperature; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011489:Protein Denaturation; D017510:Protein Folding; D016914:Ribonuclease H / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D013816:Thermodynamics; D016963:Thermus thermophilus / Q000201:enzymology*; D014961:X-Ray Diffraction
D016914:Ribonuclease H