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 mutation s is a useful strategy to generate a hyperstable mutant protein, five mutations, Gly23-->Ala, His62-->Pro, Va174-->Leu, Lys95-->Gly, and Asp 134-->His or Asn, were simultaneously introduced into Escherichia coil ribonuclease HI. The enzymatic activities of the resultant quintuple mutant proteins, 5H- and 5N-RNases HI, which have His and Asn at posit ion 134, respectively, were 35 and 55% of that of the wild-type protei n. The far-UV and near-UV CD spectra of these mutant proteins were sim ilar to those of the wild-type protein, suggesting that the mutations did not seriously affect the tertiary structure of the protein. The di fferences in the free energy change of unfolding between the wild-type and mutant proteins, Delta Delta G, were estimated by analyzing the t hermal denaturation of the proteins by CD. The SH-RNase HI protein, wh ich was slightly more stable than the SN-RNase HI, was more stable tha n the wild-type protein by 20.2 degrees C in T-m and 5.6 kcal/mol in D elta 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 th e thermal Stability and the susceptibility to chymotryptic digestion w ere nearly cumulative, and the SH-RNase HI undergoes chymotryptic dige stion at a rate that is 41 times slower than that of the wild-type pro tein. Good correlation was observed between the thermal stability and the resistance to chymotryptic digestion for all proteins examined. Th ese results suggest that the thermostabilizing mutations contribute to shift the equilibrium between the folded and unfolded states of the p rotein so that the fraction of the folded state increases.