Dissecting Contributions to the Thermostability of Pyrococcus furiosus Rubredoxin: β-Sheet Chimeras

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Abstract
The contributions to thermostability of interactions within the β-sheet region of rubredoxins (Rds) were investigated by examining proteins in which β-strand sequences of Rds from the hyperthermophilic archaeon Pyrococcus furiosus (Pf) and the mesophilic bacterium Clostridium pasteurianum (Cp) were interchanged. The thermostabilities of the chimeric Rds were assessed by monitoring the decay of the visible absorbance at 490 nm and of the far-UV CD vs time at 92 °C. The chimeric Rds Pf15|Cp47|Pf (Pf Rd residues 2−15 and 48−54 and Cp Rd residues 16−47) and Cp15|Pf47|Cp were both found to be far less thermostable than wild-type Pf Rd, indicating that neither the β-sheet residues (2−7, 10−15, and 48−53) nor the “core residues” (16−47) of Pf Rd independently confer Pf Rd-like thermostability. However, the chimeric Rd Pf47|Cp exhibits thermostability close to that of wild-type Pf Rd, suggesting that Pf Rd-like thermostability is conferred by interactions of β-sheet strands 1 and 2 (residues 2−15) together with Pf core residues. In contrast, Cp Rd β-sheet strands 1 and 2 connecting to Pf Rd core residues are thermodestabilizing in the chimera Cp15|Pf Rd. These results suggest that a global alignment which optimizes both main chain and side chain interactions between β-sheet strands and core residues is more important than a few localized interactions within the β-sheet in conferring Pf Rd-like thermostability.