title: Investigating the structural dependence of protein stabilization by amino acid substitution.
abstract: A goal of protein engineering technology is developing methods to increase protein stability. However, rational design of stable proteins is difficult because the stabilization mechanism of proteins is not fully understood. In this study, we examined the structural dependence of protein stabilization by introducing single amino acid substitution into ribonuclease H1 from the psychotropic bacterium Shewanella oneidensis MR-1 (So-RNase H1), which was our model protein. We performed saturation mutagenesis at various sites. Mutations that stabilized So-RNase H1 were screened using an RNase H-dependent temperature-sensitive Escherchia coli strain. Stabilizing mutations were identified by the suppressor mutagenesis method. This method yielded 39 stabilized mutants from 513 mutations at 27 positions. This suggested that more than 90% of mutations caused destabilization even in a psychotropic protein. However, 17 positions had stabilizing mutations, indicating that the stabilization factors were dispersed over many positions. Interestingly, the identified mutations were distributed mainly at exposed or nonconserved sites. These results provide a novel strategy for protein stabilization.
authors.affiliation: Department of Material and Life Science, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Tadokoro
authors.forename: Hirotaka
authors.initials: H
authors.lastname: Kazama
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: D019943:Amino Acid Substitution; D004926:Escherichia coli / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D055550:Protein Stability; D016914:Ribonuclease H / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D020592:Shewanella / Q000201:enzymology*
chemical_list: D016914:Ribonuclease H; C078856:ribonuclease HI