Creation of Thermostabilized Mutant for Rhodopsin Based on Statistical Thermodynamics

• Project/Area Number: 17K15099
• Research Category: Grant-in-Aid for Young Scientists (B)
• Allocation Type: Multi-year Fund
• Research Field: Biophysics
• Research Institution: Chiba University
• Principal Investigator: Satoshi Yasuda 千葉大学, 大学院理学研究院, 特任助教 (40792081)
• Project Period (FY): 2017-04-01 – 2021-03-31
• Project Status: Completed (Fiscal Year 2020)
• Budget Amount: ¥4,420,000 (Direct Cost: ¥3,400,000、Indirect Cost: ¥1,020,000); Fiscal Year 2019: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000); Fiscal Year 2018: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000); Fiscal Year 2017: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
• Keywords: サーモフィリックロドプシン / 耐熱化 / アミノ酸置換 / 統計熱力学 / 積分方程式論 / 水素結合 / エントロピー / 共溶媒効果 / 熱安定化 / 自由エネルギー関数 / 溶媒和エントロピー / 膜タンパク質 / ロドプシン / 形態計測学的アプローチ

Outline of Final Research Achievements

Although thermophilic rhodopsin (TR) and xanthorhodopsin (XR) share a high similarity in amino-acid sequence and an almost indistinguishable three-dimensional structure, TR is much more thermostable than XR. In this study, we develop the free-energy function (FEF) enabling us to evaluate the thermostability of membrane proteins and reveal the mechanism of thermostabilization of TR. Moreover, we identified the thermostabilizing mutation of TR using the FEF.

Academic Significance and Societal Importance of the Research Achievements

膜タンパク質は現在市販されている薬の約6割の標的となっているが、その安定性が低いため立体構造解析や精製標品を用いた機能解析等が非常に困難である。膜タンパク質は水中環境にある細胞内・細胞外領域と無極性環境にある膜内領域からなり、水溶性タンパク質よりも安定性を司る因子の解明が遅れている。本研究成果により解明されたTRが極めて高い耐熱性を有するメカニズムは、他の多くの不安定な膜タンパク質の安定化への有力な指針となる。またTRの耐熱化置換体は光センサー等の高機能材料としての利用が期待される。

Research Products

• [Journal Article] サーモフィリックロドプシンとキサントロドプシンの熱安定性の大きな違いに対する統計熱力学解析 (2021)
  • Author(s): 安田賢司、林智彦、村田 武士、木下正弘
  • Journal Title: アグリバイオ Volume: 4 Pages: 61-66
• [Journal Article] Elucidation of cosolvent effects thermostabilizing water-soluble and membrane proteins (2020)
  • Author(s): Yasuda Satoshi、Kazama Kazuki、Akiyama Tomoki、Kinoshita Masahiro、Murata Takeshi
  • Journal Title: Journal of Molecular Liquids Volume: 301 Pages: 112403-112403
  • DOI: 10.1016/j.molliq.2019.112403
  • Peer Reviewed
• [Journal Article] How Does a Microbial Rhodopsin RxR Realize Its Exceptionally High Thermostability with the Proton-Pumping Function Being Retained? (2020)
  • Author(s): Hayashi Tomohiko、Yasuda Satoshi、Suzuki Kano、Akiyama Tomoki、Kanehara Kanae、Kojima Keiichi、Tanabe Mikio、Kato Ryuichi、Senda Toshiya、Sudo Yuki、Murata Takeshi、Kinoshita Masahiro
  • Journal Title: The Journal of Physical Chemistry B Volume: 124 Issue: 6 Pages: 990-1000
  • DOI: 10.1021/acs.jpcb.9b10700
  • Peer Reviewed
• [Journal Article] Methodology for Further Thermostabilization of an Intrinsically Thermostable Membrane Protein Using Amino Acid Mutations with Its Original Function Being Retained (2020)
  • Author(s): Yasuda Satoshi、Akiyama Tomoki、Nemoto Sayaka、Hayashi Tomohiko、Ueta Tetsuya、Kojima Keiichi、Tsukamoto Takashi、Nagatoishi Satoru、Tsumoto Kouhei、Sudo Yuki、Kinoshita Masahiro、Murata Takeshi
  • Journal Title: Journal of Chemical Information and Modeling Volume: 60 Issue: 3 Pages: 1709-1716
  • DOI: 10.1021/acs.jcim.0c00063
  • Peer Reviewed
• [Journal Article] Theoretical identification of thermostabilizing amino acid mutations for G-protein-coupled receptors (2020)
  • Author(s): Takeshi Murata、Satoshi Yasuda、Tomohiko Hayashi、Masahiro Kinoshita
  • Journal Title: Biophysical Reviews Volume: － Issue: 2 Pages: 323-332
  • DOI: 10.1007/s12551-020-00678-5
  • Peer Reviewed
• [Journal Article] サーモフィリックロドプシンの極めて高い熱安定性に関する統計熱力学的解析 (2020)
  • Author(s): 安田賢司、林智彦、村田武士、木下正弘
  • Journal Title: 月刊「細胞」 Volume: 52 Pages: 37-41
• [Journal Article] サーモフィリックロドプシンとキサントロドプシンの熱安定性の大きな違いに対する統計熱力学解析 (2020)
  • Author(s): 安田賢司、林智彦、村田武士、木下正弘
  • Journal Title: アグリバイオ Volume: 4 Pages: 61-67
• [Journal Article] Analyses based on statistical thermodynamics for large difference between thermophilic rhodopsin and xanthorhodopsin in terms of thermostability (2019)
  • Author(s): Yasuda Satoshi、Hayashi Tomohiko、Kajiwara Yuta、Murata Takeshi、Kinoshita Masahiro
  • Journal Title: The Journal of Chemical Physics Volume: 150 Issue: 5 Pages: 055101-055101
  • DOI: 10.1063/1.5082217
  • NAID: 40022492153
  • Peer Reviewed / Open Access
• [Presentation] The influence of cosolvent on thermal stability of membrane proteins (2019)
  • Author(s): Kazuki Kazama, Satoshi Yasuda, Tomoki Akiyama, Masahiro Kinoshita, Takeshi Murata
  • Organizer: CBI学会2019年大会
• [Presentation] Analyses based on statistical thermodynamics for large difference between thermophilic rhodopsin and xanthorhodopsin in terms of thermostability (2019)
  • Author(s): Satoshi Yasuda, Tomohiko Hayashi, Yuta Kajiwara, Takeshi Murata, Masahiro Kinoshita
  • Organizer: CBI学会2019年大会
• [Presentation] Theoretical Prediction of Thermostabilizing Mutations for GPCR: Identification of Hot-Spot Residues to be Mutated Common in Class A GPCRs (2019)
  • Author(s): Satoshi Yasuda, Yuta Kajiwara, Yuki Takamuku, Nanao Suzuki, Yosuke Toyoda, Kazushi Morimoto, Ryoji Suno, So Iwata, Takuya Kobayashi, Takeshi Murata, Masahiro Kinoshita
  • Organizer: 第57回日本生物物理学会年会
• [Presentation] Further Thermo-Stabilization of Thermophilic Rhodopsin through Engineering in Intramembrane and Extramembrane Regions (2019)
  • Author(s): Tomoki Akiyama, Naoki Kunishima, Masako Hirose, Sayaka Nemoto, Satoshi Yasuda, Yuki Sudo, Takeshi Murata
  • Organizer: 第57回日本生物物理学会年会
• [Presentation] Statistical thermodynamics on the large difference between thermophilic rhodopsin and xanthorhodopsin in terms of thermostability (2018)
  • Author(s): Satoshi Yasuda, Tomohiko Hayashi, Yuta Kajiwara, Takeshi Murata, and Masahiro Kinoshita
  • Organizer: The 79th Okazaki Conference
• [Presentation] Physical origin of exceptionally high thermostability of thermophilic rhodopsin (2018)
  • Author(s): Satoshi Yasuda, Tomohiko Hayashi, Yuta Kajiwara, Takeshi Murata, Masahiro Kinoshita
  • Organizer: 第56回日本生物物理学会年会
• [Presentation] Statistical thermodynamics for remarkably high thermal stability of thermophilic rhodopsin (2018)
  • Author(s): Satoshi Yasuda
  • Organizer: The Second Symposium of Chiral Molecular Science and Technology in Chiba University -Advanced Materials Science, Biology & Nanophotonics in Chiba-
• [Presentation] Theoretical Identification of Hot-Spot Residues to be Mutated Common in G Protein-Coupled Receptors of Class A (2018)
  • Author(s): Satoshi Yasuda
  • Organizer: Biophysical Society 62nd Annual Meeting
  • Int'l Joint Research
• [Presentation] 統計熱力学に基づいたG蛋白質共役型受容体耐熱化アミノ酸変異体の理論的予測 (2017)
  • Author(s): 安田賢司
  • Organizer: 第17回日本蛋白質科学会年会
• [Presentation] 置換により多くのClass AのGタンパク質共役型を安定化するアミノ酸残基の理論的決定 (2017)
  • Author(s): 安田賢司
  • Organizer: 第５５回日本生物物理学会年会
• [Presentation] サーモフィリックロドプシンの極めて高い熱安定性に対する統計熱力学的解析 (2017)
  • Author(s): 安田賢司
  • Organizer: 日本生体エネルギー研究会第43回討論会