title: Adaptation of a hyperthermophilic group II chaperonin to relatively moderate temperatures.
abstract: Group II chaperonins exist in archaea and the eukaryotic cytosol, and mediate protein folding in an ATP-dependent manner. We have been studying the reaction mechanism of group II chaperonins using alpha chaperonin, the recombinant chaperonin alpha subunit homo-oligomer from a hyperthermophilic archaeon, Thermococcus sp. strain KS-1 (T. KS-1). Although the high stability and activity of T. KS-1 alpha chaperonin provided advantages for our study, its high thermophilicity caused the difficulty in using various analytical methods. To resolve this problem, we tried to adapt T. KS-1 alpha chaperonin to moderate temperatures by mutations. The comparison of amino acid sequences between 26 thermophilic and 17 mesophilic chaperonins showed that three amino acid replacements are likely responsible for the difference of their optimal temperatures. We introduced three single mutations and also their double combinations into T. KS-1 alpha chaperonin. Among them, K323R single mutant exhibited the improvements of the folding activity and the ATP-dependent conformational change ability at lower temperatures, such as 50 degrees C and 40 degrees C. Since K323 may secure helix 12 in the closed conformation by interacting with D198, the replacement of Lys to Arg likely induced the higher mobility of the built-in lid, resulting in the higher activity at relatively low temperatures.
authors.affiliation: Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei-shi, Tokyo 184-8588, Japan.
authors.forename: Taro
authors.initials: T
authors.lastname: Kanzaki
authors.forename: Shuzo
authors.initials: S
authors.lastname: Ushioku
authors.forename: Ayumi
authors.initials: A
authors.lastname: Nakagawa
authors.forename: Toshihiko
authors.initials: T
authors.lastname: Oka
authors.forename: Kazunobu
authors.initials: K
authors.lastname: Takahashi
authors.forename: Takashi
authors.initials: T
authors.lastname: Nakamura
authors.forename: Kunihiro
authors.initials: K
authors.lastname: Kuwajima
authors.forename: Akihiko
authors.initials: A
authors.lastname: Yamagishi
authors.forename: Masafumi
authors.initials: M
authors.lastname: Yohda
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D056385:Group II Chaperonins / Q000235:genetics*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D020125:Mutation, Missense / Q000235:genetics; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding; D011994:Recombinant Proteins / Q000235:genetics*; D017422:Sequence Analysis, DNA; D013696:Temperature; D019712:Thermococcus / Q000737:chemistry*
chemical_list: D017931:DNA Primers; D011994:Recombinant Proteins; D056385:Group II Chaperonins