HIGH-RESISTANCE OF ESCHERICHIA-COLI RIBONUCLEASE HI VARIANT WITH QUINTUPLE THERMOSTABILIZING MUTATIONS TO THERMAL-DENATURATION, ACID DENATURATION, AND PROTEOLYTIC DEGRADATION

TO test whether the combination of multiple thermostabilizing mutations is a useful strategy to generate a hyperstable mutant protein, five mutations, Gly23-->Ala, His62-->Pro, Va174-->Leu, Lys95-->Gly, and Asp134-->His or Asn, were simultaneously introduced into Escherichia coli ribonuclease HI. The enzymatic activities of the resultant quintuple mutant proteins, 5H- and 5N-RNases HI, which have His and Asn at position 134, respectively, were 35 and 55% of that of the wild-type protein. The far-UV and near-UV CD spectra of these mutant proteins were similar to those of the wild-type protein, suggesting that the mutations did not seriously affect the tertiary structure of the protein. The differences in the free energy change of unfolding between the wild-type and mutant proteins, Delta Delta G, were estimated by analyzing the thermal denaturation of the proteins by CD. The SH-RNase HI protein, which was slightly more stable than the SN-RNase HI, was more stable than the wild-type protein by 20.2 degrees C in T-m and 5.6 kcal/mol in Delta G at pH 5.5. In addition, the SH-RNase HI was highly resistant to proteolysis and acid denaturation. The effects of each mutation on the thermal Stability and the susceptibility to chymotryptic digestion were nearly cumulative, and the SH-RNase HI undergoes chymotryptic digestion at a rate that is 41 times slower than that of the wild-type protein. Good correlation was observed between the thermal stability and the resistance to chymotryptic digestion for all proteins examined. These results suggest that the thermostabilizing mutations contribute to shift the equilibrium between the folded and unfolded states of the protein so that the fraction of the folded state increases.