2020 Fiscal Year Final Research Report
Molecular Design and Functional Regulation of Integrated Membrane-bound Protein Using Nono-disc
| Project/Area Number |
19K22193
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| Research Category |
Grant-in-Aid for Challenging Research (Exploratory)
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| Allocation Type | Multi-year Fund |
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| Review Section |
Medium-sized Section 34:Inorganic/coordination chemistry, analytical chemistry, and related fields
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| Research Institution | Hokkaido University |
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Principal Investigator |
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| Co-Investigator(Kenkyū-buntansha) |
内田 毅 北海道大学, 理学研究院, 准教授 (30343742)
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| Project Period (FY) |
2019-06-28 – 2021-03-31
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| Keywords | ナノディスク / 膜結合蛋白質 / 電極反応 / シトクロム酸化酵素 / ハロロドプシン / 光駆動塩素イオンポンプ |
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| Outline of Final Research Achievements |
We focused on nonodisc-reconstituted halorhodopsin (HR), pumping chloride ion into the cell in response to light, and bacterial cytochrome oxidase (cbb3), promoting four-electron reduction of molecular oxygen in the respiratory chain. Their structures and enzymatic activities were investigated by various kinds of spectroscopies, and the applications of these nanodisc-reconstituted proteins were examined. To facilitate the effective chloride pumping in HR, the interactions between HRs, effects of charges on the membrane, and flexibility of the membrane to tolerate the conformational changes associated with the chloride binding and releasing were found to be essential. We successfully immobilized cbb3 on the electrode and found that immobilized cbb3 can electrochemically mediate the four-electron reduction of molecular oxygen on the electrode. Further experiments are required to improve the efficiency of the reduction of molecular oxygen by using the nanodisc-reconstituted enzyme.
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| Free Research Field |
生物無機化学
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| Academic Significance and Societal Importance of the Research Achievements |
細胞内で重要な機能を果たしている膜結合蛋白質は、精製蛋白質として細胞外で機能させる場合、これまで界面活性剤による可溶化が用いられてきたが、このような手法では膜結合蛋白質が膜との相互作用によって維持してきた構造安定性や結合配向性が失われ、細胞内と同様な機能を発揮できない場合が多い。本研究では、安定な円盤状脂質膜であるナノディスクに膜結合蛋白質を結合させることで、膜結合による蛋白質構造や機能への影響を明らかにすることができ、さらに酸化酵素が電極上での酸素分子の水への還元反応の触媒として機能することを確認できた。以上の成果は、膜蛋白質の機能分子としての応用を考えるうえでの指針となることが期待される。
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