title: Protein thermostabilization requires a fine-tuned placement of surface-charged residues.
abstract: Using the information from the genome projects, recent comparative studies of thermostable proteins have revealed a certain trend of amino acid composition in which polar residues are scarce and charged residues are rich on the protein surface. To clarify experimentally the effect of the amino acid composition of surface residues on the thermostability of Escherichia coli Ribonuclease HI (RNase HI), we constructed six variants in which five to eleven polar residues were replaced by charged residues (5C, 7Ca, 7Cb, 9Ca, 9Cb and 11C). The thermal denaturation experiments indicated that all of the variant proteins are 3.2-10.1 degrees C in Tm less stable than the wild proteins. The crystal structures of resultant protein variants 7Ca, 7Cb, 9Ca and 11C closely resemble that of E. coli RNase HI in their global fold, and several different hydrogen bonding and ion-pair interactions are formed by the mutations. Comparison of the crystal structures of these variant proteins with that of E. coli RNase HI reveals that thermal destabilization is apparently related to electrostatic repulsion of the charged residues with neighbours. This result suggests that charged residues of natural thermostable proteins are strictly posted on the surface with optimal interactions and without repulsive interactions.
authors.affiliation: Department of Material and Life Science, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Dong-Ju
authors.initials: DJ
authors.lastname: You
authors.forename: Satoshi
authors.initials: S
authors.lastname: Fukuchi
authors.forename: Ken
authors.initials: K
authors.lastname: Nishikawa
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Koga
authors.forename: Kazufumi
authors.initials: K
authors.lastname: Takano
authors.forename: Shigenori
authors.initials: S
authors.lastname: Kanaya
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000596:Amino Acids / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D014644:Genetic Variation; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016914:Ribonuclease H / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D055672:Static Electricity; D013499:Surface Properties; D013816:Thermodynamics
chemical_list: D000596:Amino Acids; D016914:Ribonuclease H; C078856:ribonuclease HI