Stabilization of Escherichia coli ribonuclease HI by strategic replacement of amino acid residues with those from the thermophilic counterpart.
Thermus thermophilus ribonuclease H is exceptionally stable against thermal and guanidine hydrochloride denaturations as compared to Escherichia coli ribonuclease HI (Kanaya, S., and Itaya, M. (1992) J. Biol. Chem. 267, 10184-10192). The identity in the amino acid sequences of these enzymes is 52%. As an initial step to elucidate the stabilization mechanism of the thermophilic RNase H, we examined whether certain regions in its amino acid sequence confer the thermostability. A variety of mutant proteins of E. coli RNase HI were constructed and analyzed for protein stability. In these mutant proteins, amino acid sequences in loops or terminal regions were systematically replaced with the corresponding sequences from T. thermophilus RNase H. Of the nine regions examined, replacement of the amino acid sequence in each of four regions (R4-R7) resulted in an increase in protein stability. Simultaneous replacements of these amino acid sequences revealed that the effect of each replacement on protein stability is independent of each other and cumulative. Replacement of all four regions (R4-R7) gave the most stable mutant protein. The temperature of the midpoint of the transition in the thermal unfolding curve and the free energy change of unfolding in the absence of denaturant of this mutant protein were increased by 16.7 degrees C and 3.66 kcal/mol, respectively, as compared to those of E. coli RNase HI. These results suggest that individual local interactions contribute to the stability of thermophilic proteins in an independent manner, rather than in a cooperative manner.
Kimura
S
S
Protein Engineering Research Institute, Osaka, Japan.
Nakamura
H
H
Hashimoto
T
T
Oobatake
M
M
Kanaya
S
S
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics; D001483:Base Sequence; D002942:Circular Dichroism; D004277:DNA, Single-Stranded; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006358:Hot Temperature; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010957:Plasmids; D011489:Protein Denaturation; D017510:Protein Folding; D016914:Ribonuclease H / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D016963:Thermus thermophilus / Q000201:enzymology*
D000596:Amino Acids; D004277:DNA, Single-Stranded; D016914:Ribonuclease H