Stabilization of ribonuclease HI from Thermus thermophilus HB8 by the spontaneous formation of an intramolecular disulfide bond

TO identify factors that contribute to the thermal stability of ribonuclease HI (RNase HI) from Thermus thermophilus HB8, protein variants with a series of carboxyl-terminal truncations and Cys Ala mutations were constructed, and their thermal denaturations were analyzed by CD. The results indicate that Cys(41) and Cys(149) contribute to the protein stability, probably through the formation of a disulfide bond. Peptide mapping analysis for the mutant protein with only two cysteine residues, at positions 41 and 149, indicated that this disulfide bond is partially formed in a protein purified from Escherichia coli in the absence of a reducing reagent but is fully formed in a thermally denatured protein. These results suggest that the thermal stability of T. thermophilus RNase HI, determined in the absence of a reducing reagent, reflects that of an oxidized form of the protein. Comparison of the thermal stabilities and the enzymatic activities of the wild-type and truncated proteins, determined in the presence and absence of a reducing reagent, indicates that the formation of this disulfide bond increases the thermal stability of the protein by 6-7 degrees C in T-m and similar to 3 kcal/mol in Delta G without seriously affecting the enzymatic activity. Since T. thermophilus RNase HT is present in a reducing environment in cells, this disulfide bond probably is not formed in vivo but is spontaneously formed in vitro in the absence of a reducing reagent.