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2019 Fiscal Year Final Research Report

Detection of the physical interaction between rotor and stator essential for torque generation in the bacterial flagellar motor

Research Project

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Project/Area Number 18K19293
Research Category

Grant-in-Aid for Challenging Research (Exploratory)

Allocation TypeMulti-year Fund
Review Section Medium-sized Section 43:Biology at molecular to cellular levels, and related fields
Research InstitutionNagoya University

Principal Investigator

Kojima Seiji  名古屋大学, 理学研究科, 准教授 (70420362)

Project Period (FY) 2018-06-29 – 2020-03-31
Keywords細菌べん毛モーター / 回転子 / 固定子 / 部位特異的光架橋 / MotA / PomA / FliG
Outline of Final Research Achievements

Bacterial flagellar motor is a rotary nanomachine driven by the ion-motive force across the cytoplasmic membrane. Rotational force is generated by the rotor-stator interaction that couples to ion conduction through the stator complex. Since the rotor-stator interaction occurs very transient and rapidly, it was not detected physically or biochemically in protein level. Here we employed the in vivo photo-crosslink method to capture this rapid interaction. In this approach, the photo-reactive but non-natural amino acid (pBPA) was site-directly incorporated into the rotor or stator protein respectively (FliG or MotA/PomA) in Eschericha coli cell. After the UV flash irradiation to the cell, the photo-crosslinked protein was detected by the SDS-PAGE followed by the immunoblotting. Using this method, we could detect for the first time FliG-MotA or FliG-PomA interactions between the residues reported genetically for the electrostatic interactions.

Free Research Field

生化学・分子生物学・生物物理学

Academic Significance and Societal Importance of the Research Achievements

細菌べん毛モーターの回転機構を理解するためには、回転力発生の核となる回転子-固定子間相互作用の実態を明らかにする必要がある。しかし高速回転するモーターにおいて短時間かつ連続的に生じるこの相互作用は、これまでタンパク質レベルで検出されていなかった。本研究では光を用いて瞬時に相互作用を捉える光架橋法を用いて、初めて固定子と回転子間のタンパク質間相互作用を検出することに成功した。本研究で見出した相互作用残基は、遺伝学的解析で同定されていた静電相互作用に関与する残基であり、今後、最近得られた固定子と回転子タンパク質の立体構造情報と合わせた解析により、相互作用界面の実態を明らかにすることが期待できる。

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Published: 2021-02-19  

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