title: Stabilization of ribonuclease HI from Thermus thermophilus HB8 by the spontaneous formation of an intramolecular disulfide bond.
abstract: To identify factors that contribute to the thermal stability of ribonuclease HI (RNase HI) from Thermus thermophilus HB8, protein variants with a series of carboxyl-terminal truncations and Cys --> Ala mutations were constructed, and their thermal denaturations were analyzed by CD. The results indicate that Cys41 and Cys149 contribute to the protein stability, probably through the formation of a disulfide bond. Peptide mapping analysis for the mutant protein with only two cysteine residues, at positions 41 and 149, indicated that this disulfide bond is partially formed in a protein purified from Escherichia coli in the absence of a reducing reagent but is fully formed in a thermally denatured protein. These results suggest that the thermal stability of T. thermophilus RNase HI, determined in the absence of a reducing reagent, reflects that of an oxidized form of the protein. Comparison of the thermal stabilities and the enzymatic activities of the wild-type and truncated proteins, determined in the presence and absence of a reducing reagent, indicates that the formation of this disulfide bond increases the thermal stability of the protein by 6-7 degreesC in Tm and approximately 3 kcal/mol in DeltaG without seriously affecting the enzymatic activity. Since T. thermophilus RNase HI is present in a reducing environment in cells, this disulfide bond probably is not formed in vivo but is spontaneously formed in vitro in the absence of a reducing reagent.
authors.affiliation: Department of Material and Life Science, Graduate School of Engineering, Osaka University, Japan.
authors.forename: N
authors.initials: N
authors.lastname: Hirano
authors.forename: M
authors.initials: M
authors.lastname: Haruki
authors.forename: M
authors.initials: M
authors.lastname: Morikawa
authors.forename: S
authors.initials: S
authors.lastname: Kanaya
mesh_terms: D000595:Amino Acid Sequence; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000235:genetics; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism; D004789:Enzyme Activation / Q000235:genetics; D004795:Enzyme Stability / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000235:genetics / Q000302:isolation & purification; D010449:Peptide Mapping; D011489:Protein Denaturation; D019163:Reducing Agents; D016914:Ribonuclease H / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016963:Thermus thermophilus / Q000201:enzymology*
chemical_list: D004220:Disulfides; D010446:Peptide Fragments; D019163:Reducing Agents; D016914:Ribonuclease H; C078856:ribonuclease HI; D003545:Cysteine