CONFORMATIONAL STABILITIES OF ESCHERICHIA-COLI RNASE HI VARIANTS WITHA SERIES OF AMINO-ACID SUBSTITUTIONS AT A CAVITY WITHIN THE HYDROPHOBIC CORE

Escherichia coli ribonuclease HI has a cavity within the hydrophobic c ore, Two core residues, Ala(52) and Val(74), resided at both ends of t his cavity, We have constructed a series of single mutant proteins at Ala(52), and double mutant proteins, in which Ala(52) was replaced by Gly, Val, Ile, Leu, or Phe, and Val(74) was replaced by Ala or Leu, Al l of these mutant proteins, except for A52W, A52R, and A52G/V74A, were overproduced and purified, Measurement of the thermal denaturations o f the proteins at pH 3.2 by CD suggests that the cavity is large enoug h to accommodate three methyl or methylene groups without creating ser ious strains, A correlation was observed between the protein stability and the hydrophobicity of the substituted residue, As a result, a num ber of the mutant proteins were more stable than the wild-type protein , The stabilities of the mutant proteins with charged or extremely bul ky residues at the cavity were lower than those expected from the hydr ophobicities of the substituted residues, suggesting that considerable strains are created at the mutation sites in these mutant proteins, H owever, examination of the far- and near-UV GD spectra and the enzymat ic activities suggest that all of the mutant proteins have structures similar to that of the wild-type protein, These results suggest that t he cavity in the hydrophobic core off. coli RNase HI is conformational ly fairly stable, This may be the reason why the cavity-filling mutati ons effectively increase the thermal stability of this protein.