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2020 Fiscal Year Research-status Report

Structural Dynamics of ABC Transporter MsbA during Functional Activity

Research Project

Project/Area Number 19K06581
Research InstitutionKanazawa University

Principal Investigator

NGO XUANKIEN  金沢大学, ナノ生命科学研究所, 特任助教 (60778190)

Project Period (FY) 2019-04-01 – 2022-03-31
KeywordsABC transporters / MsbA / lipid A / lipopolysaccharide / proteoliposomes / giant proteovesicles / HS-AFM / STED confocal microsopy
Outline of Annual Research Achievements

The objectives of this project are (i) To clarify how the ATPase activity of MsbA is affected by different substrates and a proton gradient; (ii) To reveal the dynamic conformational changes of MsbA during functional activity and to determine its rate as a function of [ATP] and [substrates]; To clarify whether or not conformational changes of MsbA can also be induced by an electrochemical proton gradient established between both sides of the lipid membrane, in a way similar to the case of ATPase cycle; (iv) To clarify how ATP and a proton gradient show a synergistic effect on the size and rate of conformational changes of MsbA. Overall, I have successfully (i) prepared detergent-solubilized MsbA and MsbA incorporated into lipid membranes of small and giant unilamellar vesicles, (ii) real-time HS-AFM imaging of the structures and dynamics of detergent-soluble MsbA and MsbA incorporated into lipid membranes of liposomes under different nucleotide conditions (ATP, ADP, ATPγS, and nucleotide-free) using HS-AFM. ABC transporter, (iii) analyze and compare the data obtained by HS-AFM with the data acquired by a coarse-grained simulation, and vice versa, to explore the molecular mechanism underlying the ATP consuming molecular machines such as the relationship between the dynamic structural changes in NBDs and TMDs of MsbA with the lipid transport activity across the membrane.

Current Status of Research Progress
Current Status of Research Progress

3: Progress in research has been slightly delayed.

Reason

In this project, I have addressed several important scientific questions such as (i) To analyze dynamic conformational changes at a high spatiotemporal resolution of detergent-soluble MsbA and MsbA incorporated into lipid membranes using HS-AFM, and determine its rate as a function of [nucleotides] and [substrates]; (ii)analyze the data acquired by HS-AFM and compared them to data obtained by a coarse-grained simulation, and vice versa, to explore the detailed structural dynamics of MsbA. However, we have not succeeded to capture in real-time the “lipid transport activity” of MsbA using HS-AFM. For this challenging purpose, we currently develop a novel assay system to form the suspended lipid membranes, furnishing a freestanding membrane incorporating MsbA transporters during functional activities, for HS-AFM observation. Indeed, we have successfully screened, fabricated, and optimized several nanoporous films for this purpose. The nanoporous film is equipped with a glass chamber, enabling the microliter solution to come in and out circularly, controlled by a micro-syringe push-pull pump. This configuration allows me to examine free conformational changes and lipid (lipid A or LPS) transport activities of MsbA incorporated in the suspended membrane using HS-AFM.

Strategy for Future Research Activity

In FY2021, I will accomplish the following goals (i) analyze structural dynamics and transport activity of MsbA incorporated into the suspended lipid membrane using HS-AFM; (ii) analyze the HS-AFM data and compare them with the data obtained by a coarse-grained simulation, and vice versa, for understanding the detailed molecular operation of MsbA; (iii) publish the scientific data to the proper scientific journals.

Remarks

We developed several assay systems enabling us to incorporate the bacterial ABC transporter, MsbA, into the suspended lipid membranes-like an artificial cytoplasmic membrane

  • Research Products

    (3 results)

All 2022 2021

All Journal Article (3 results) (of which Int'l Joint Research: 2 results,  Peer Reviewed: 2 results,  Open Access: 3 results)

  • [Journal Article] Actin binding domain of Rng2 sparsely bound on F-actin strongly inhibits actin movement on myosin II2022

    • Author(s)
      Hayakawa Yuuki、Takaine Masak、Ngo Kien Xuan、Imai Taiga、Yamada Masafumi D.、Behjat Arash Badami、Umeda Kenichi、Hirose Keiko、Yurtsever Ayhan、Kodera Noriyuki、Tokuraku Kiyotaka、Numata Osamu、Fukuma Takeshi、Ando Toshio、Nakano Kentaro、Uyeda Taro Q.P.
    • Journal Title

      bioRxiv

      Volume: 000 Pages: 000

    • DOI

      10.1101/2020.04.14.041046

    • Open Access / Int'l Joint Research
  • [Journal Article] GTP-dependent formation of straight tubulin oligomers leads to microtubule nucleation2021

    • Author(s)
      Ayukawa Rie、Iwata Seigo、Imai Hiroshi、Kamimura Shinji、Hayashi Masahito、Ngo Kien Xuan、Minoura Itsushi、Uchimura Seiichi、Makino Tsukasa、Shirouzu Mikako、Shigematsu Hideki、Sekimoto Ken、Gigant Benoit、Muto Etsuko
    • Journal Title

      Journal of Cell Biology

      Volume: 220 Pages: 1-19

    • DOI

      10.1083/jcb.202007033

    • Peer Reviewed / Open Access / Int'l Joint Research
  • [Journal Article] Unraveling the host-selective toxic interaction of cassiicolin with lipid membranes and its cytotoxicity2021

    • Author(s)
      Ngo Kien Xuan、Quoc Nguyen Bao、Nguyen Phuong Doan N.、Furusho Hirotoshi、Miyata Makoto、Shimonaka Tomomi、Chau Nguyen Ngoc Bao、Vinh Nguyen Phuong、Nghia Nguyen Anh、Mohammed Tareg Omer、Ichikawa Takehiko、Kodera Noriyuki、Konno Hiroki、Fukuma Takeshi、Ando Toshio
    • Journal Title

      bioRxiv

      Volume: 0 Pages: 0

    • DOI

      10.1101/2021.04.06.438527

    • Peer Reviewed / Open Access

URL: 

Published: 2021-12-27  

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