Structural basis for thermostability in aporubredoxins from Pyrococcus furiosus and Clostridium pasteurianum

The structures of apo- and holorubredoxins from Pyrococcus furiosus (PfRd) and Clostridium pasteurianum (CpRd) have been investigated and compared usi ng residual dipolar couplings to probe the origin of thermostability. In th e native, metal (Fe or Zn) containing form, both proteins can maintain nati ve structure at very high temperatures (>70 degreesC) for extended periods of time. Significant changes in either structure or backbone dynamics betwe en 25 and 70 degreesC are not apparent for either protein. A kinetic differ ence with respect to metal loss is observed as in previous studies, but the extreme stability of both proteins in the presence of metal makes thermody namic differences difficult to monitor. In the absence of metal, however, a largely reversible thermal denaturation can be monitored, and a comparison of the two apoproteins can offer insights into the origin of stability. Be low denaturation temperatures apo-PfRd is found to have a structure nearly identical to that of the native hole form, except immediately adjacent to t he metal binding site. In contrast, apo-CpRd is found to have a structure d istinctly different from that of its hole form at low temperatures. This st ructure is rapidly lost upon heating, unfolding at approximately 40 degrees C. A PfRd mutant with the hydrophobic cope mutated to match that of CpRd sh ows no change in thermostability in the metal-free state. A metal-free chim era with residues 1-15 of CpRd and the remaining 38 residues of PfRd is sev erely destabilized and is unfolded at 25 degreesC. Hence, the hydrophobic c ore does not seem to be the key determinant of thermostability; instead, da ta point to the hydrogen bond network centered on the first 15 residues or the interaction of these 15 residues with other parts of the protein as a p ossible contributor to the thermostability.