Thermostabilization of Escherichia coli ribonuclease HI by replacing left-handed helical Lys95 with Gly or Asn.
From the systematic replacements of amino acid residues of Escherichia coli ribonuclease HI with those of its thermophilic counterpart, the basic protrusion domain including region 6 (R6) from residues 91 to 95 was found to increase the structural stability of the mutant protein (Kimura, S., Nakamura, H., Hashimoto, T., Oobatake, M., and Kanaya, S. (1992) J. Biol. Chem. 267, 21535-21542). Further mutagenesis concentrating in the R6 region has revealed that replacements of Lys95 at the left-handed structure with Gly or Asn essentially enhances the protein stability. Gly and Asn substitutions stabilize the protein up to 1.9 kcal/mol and 0.9 kcal/mol in the free energy changes of unfolding, respectively. We propose that the amino acid substitution of left-handed non-Gly residue with Gly or Asn residue can be used as one of the general strategies to enhance protein stability, when such a non-Gly residue itself does not seriously contribute to protein stability.
Kimura
S
S
Protein Engineering Research Institute, Osaka, Japan.
Kanaya
S
S
Nakamura
H
H
D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000201:enzymology; D006358:Hot Temperature; D008969:Molecular Sequence Data; D011489:Protein Denaturation; D016914:Ribonuclease H / Q000737:chemistry*; D013329:Structure-Activity Relationship
D016914:Ribonuclease H