| 研究実績の概要 |
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.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
3: やや遅れている
理由
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.
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| 今後の研究の推進方策 |
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.
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