title: Protein stabilization utilizing a redefined codon.
abstract: Recent advances have fundamentally changed the ways in which synthetic amino acids are incorporated into proteins, enabling their efficient and multiple-site incorporation, in addition to the 20 canonical amino acids. This development provides opportunities for fresh approaches toward addressing fundamental problems in bioengineering. In the present study, we showed that the structural stability of proteins can be enhanced by integrating bulky halogenated amino acids at multiple selected sites. Glutathione S-transferase was thus stabilized significantly (by 5.2 and 5.6 kcal/mol) with 3-chloro- and 3-bromo-l-tyrosines, respectively, incorporated at seven selected sites. X-ray crystallographic analyses revealed that the bulky halogen moieties filled internal spaces within the molecules, and formed non-canonical stabilizing interactions with the neighboring residues. This new mechanism for protein stabilization is quite simple and applicable to a wide range of proteins, as demonstrated by the rapid stabilization of the industrially relevant azoreductase.
authors.affiliation: 1] Division of Structural and Synthetic Biology, RIKEN Center for Life Science Technologies, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan [2] RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan.
authors.forename: Kazumasa
authors.initials: K
authors.lastname: Ohtake
authors.affiliation: 1] Division of Structural and Synthetic Biology, RIKEN Center for Life Science Technologies, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan [2] RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Yamaguchi
authors.affiliation: 1] Division of Structural and Synthetic Biology, RIKEN Center for Life Science Technologies, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan [2] RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan.
authors.forename: Takahito
authors.initials: T
authors.lastname: Mukai
authors.affiliation: Department of Chemical Biology and Applied Chemistry, College of Engineering, Nihon University, Koriyama, Fukushima 963-8642, Japan.
authors.forename: Hiroki
authors.initials: H
authors.lastname: Kashimura
authors.affiliation: Department of Chemical Biology and Applied Chemistry, College of Engineering, Nihon University, Koriyama, Fukushima 963-8642, Japan.
authors.forename: Nobutaka
authors.initials: N
authors.lastname: Hirano
authors.affiliation: Department of Chemical Biology and Applied Chemistry, College of Engineering, Nihon University, Koriyama, Fukushima 963-8642, Japan.
authors.forename: Mitsuru
authors.initials: M
authors.lastname: Haruki
authors.affiliation: Department of Chemical Biology and Applied Chemistry, College of Engineering, Nihon University, Koriyama, Fukushima 963-8642, Japan.
authors.forename: Sosuke
authors.initials: S
authors.lastname: Kohashi
authors.affiliation: Department of Chemical Biology and Applied Chemistry, College of Engineering, Nihon University, Koriyama, Fukushima 963-8642, Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Yamagishi
authors.affiliation: Biomedical Engineering Research Organization, Tohoku University, Aoba-ku, Sendai 980-8575, Japan.
authors.forename: Kazutaka
authors.initials: K
authors.lastname: Murayama
authors.affiliation: 1] Division of Structural and Synthetic Biology, RIKEN Center for Life Science Technologies, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan [2] RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan.
authors.forename: Yuri
authors.initials: Y
authors.lastname: Tomabechi
authors.affiliation: RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Itagaki
authors.affiliation: 1] Division of Structural and Synthetic Biology, RIKEN Center for Life Science Technologies, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan [2] RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan.
authors.forename: Ryogo
authors.initials: R
authors.lastname: Akasaka
authors.affiliation: 1] RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan [2] RIKEN Structural Biology Laboratory, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan.
authors.forename: Masahito
authors.initials: M
authors.lastname: Kawazoe
authors.affiliation: 1] Division of Structural and Synthetic Biology, RIKEN Center for Life Science Technologies, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan [2] RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan.
authors.forename: Chie
authors.initials: C
authors.lastname: Takemoto
authors.affiliation: 1] Division of Structural and Synthetic Biology, RIKEN Center for Life Science Technologies, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan [2] RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan.
authors.forename: Mikako
authors.initials: M
authors.lastname: Shirouzu
authors.affiliation: 1] RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan [2] RIKEN Structural Biology Laboratory, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan.
authors.forename: Shigeyuki
authors.initials: S
authors.lastname: Yokoyama
authors.affiliation: 1] Division of Structural and Synthetic Biology, RIKEN Center for Life Science Technologies, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan [2] RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan.
authors.forename: Kensaku
authors.initials: K
authors.lastname: Sakamoto