DISSECTING CONTRIBUTIONS TO THE THERMOSTABILITY OF PYROCOCCUS-FURIOSUS RUBREDOXIN - BETA-SHEET CHIMERAS

The contributions to thermostability of interactions within the beta-s heet region of rubredoxins (Rds) were investigated by examining protei ns in which beta-strand sequences of Rds from the hyperthermophilic ar chaeon Pyrococcus furiosus (Pf) and the mesophilic bacterium Clostridi um pasteurianrum (Cp) were interchanged. The thermostabilities of the chimeric Rds were assessed by monitoring the decay of the visible abso rbance at 490 nm and of the far-UV CD vs time at 92 degrees C. The chi meric Rds Pf15\Cp47\Pf (Pf Rd residues 2-15 and 48-54 and Cp Rd residu es 16-47) and Cp15\Pf47\Cp were both found to be far less thermostable than wild-type Pf Rd. indicating that neither the beta-sheet residues (2-7, 10-15, and 48-53) nor the ''core residues'' (16-47) of Pf Rd in dependently confer Pf Rd-like thermostability. However, the chimeric R d Pf47\Cp exhibits thermostability close to that of wild-type Pf Rd, s uggesting that Pf Rd-like thermostability is conferred by interactions of beta-sheet strands 1 and 2 (residues 2-15) together with Pf core r esidues. In contrast, Cp Rd beta-sheet strands 1 and 2 connecting to P f Rd core residues are thermodestabilizing in the chimera Cp 15\Pf Rd. These results suggest that a global alignment which optimizes both ma in chain and side chain interactions between beta-sheet strands and co re residues is more important than a few localized interactions within the beta-sheet in conferring Pf Rd-like thermostability.