2021 Fiscal Year Final Research Report
Redox sensing by membrane proteins via cysteine residues in the TM region as studied by using solution NMR
| Project/Area Number |
19K07040
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Multi-year Fund |
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| Section | 一般 |
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| Review Section |
Basic Section 47020:Pharmaceutical analytical chemistry and physicochemistry-related
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| Research Institution | The University of Tokyo (2021)
National Institute of Advanced Industrial Science and Technology (2019-2020) |
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Principal Investigator |
Tokunaga Yuji 東京大学, 大学院薬学系研究科(薬学部), 助教 (80713354)
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| Co-Investigator(Kenkyū-buntansha) |
坂倉 正義 横浜市立大学, 生命医科学研究科, 助教 (20334336)
鴫 直樹 国立研究開発法人産業技術総合研究所, 生命工学領域, 主任研究員 (20392623)
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| Project Period (FY) |
2019-04-01 – 2022-03-31
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| Keywords | タンパク質 / システイン残基 / 過硫化 / tRNA修飾 / 核磁気共鳴 / 高分子量観測 |
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| Outline of Final Research Achievements |
We developed an NMR technique, 15N-direct detection CRINEPT, which enables observation of backbone amide signals of non-deuterated high molecular weight proteins with an unprecedentedly high sensitivity. By using 15N CRINEPT, we successfuly observed amide 1H-15N resonances from more than 80% Ala residues of an analogue of a therapeutic monoclonal antibody in formulation at a storage temperature of 4 deg. This technique is also fundamental to the analyses of redox sensing mechanisms by membrane proteins reconstituted in a lipid bilayer.
We also investigated the structural change of an E. coli protein, TusE, which works as a member of sulfur transfer cascade from L-cysteine to tRNA. Persulfidation of TusE on Cys108 residue, where the cargo sulfur atom is attached to the sidechain of Cys108 to form the -SSH group, induced substantial structural change of TusE, leading to protection of the sulfur atom from passive oxidation for high-efficiency sulfer transfer.
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| Free Research Field |
構造生物学
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| Academic Significance and Societal Importance of the Research Achievements |
本研究で開発した15N CRINEPT法は、溶液NMR法の最大の課題である高分子量限界を大幅に改善するとともに、従来は解析が困難であった重水素化できないタンパク質にも適用範囲を持つため、膜タンパク質のレドックス感知機構の解明のみならず、基礎研究および創薬分野に幅広く波及効果をもたらす極めて普遍性の高い技術的進歩として有意義である。
TusEを対象としたNMR解析から明らかとされた硫化に伴う合目的的な構造変化は、化学的な安定性の低い硫黄修飾を介した情報伝達に対して頑強性をもたらす機構と捉えることができ、近年脚光を浴び始めた生体内の超硫黄分子群の制御を研究する上での先駆的な知見となる。
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