2019 Fiscal Year Research-status Report
Structural Dynamics of ABC Transporter MsbA during Functional Activity
| Project/Area Number |
19K06581
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| Research Institution | Kanazawa University |
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Principal Investigator |
NGO XUANKIEN 金沢大学, ナノ生命科学研究所, 特任助教 (60778190)
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| Project Period (FY) |
2019-04-01 – 2022-03-31
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| Keywords | ABC transporters / MsbA / BtuCDF / HS-AFM / Lipid A / Vitamin B12 / Proteoliposomes / Nanodiscs |
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| Outline of Annual Research Achievements |
We succeeded in (i) preparation and biochemical characterization of bacterial ABC transporters (i.e. MsbA and BtuCDF); and (ii) HS-AFM imaging a whole structure of detergent-solubilized MsbA bound different nucleotides; (iii) fabricating "nanopatterned surfaces" to form stably the freestanding lipid bilayer incorporating ABC transporter, MsbA, for HS-AFM imaging; (iv) preparing MsbA in the forms of proteoliposomes and nanodiscs and imaging structure and dynamics of NBDs and TMDs of MsbA embedded in these lipid membranes under physiological conditions. We currently collect and analyze more data of MsbA in lipid membrane bound different nucleotides using HS-AFM. The results have been presented in several national and international scientific conferences in FY2019.
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| Current Status of Research Progress |
Current Status of Research Progress
1: Research has progressed more than it was originally planned.
Reason
My implementation plans in FY2019 are (i) incorporation of MsbA into lipid membranes in the forms of MsbA proteoliposomes (MsbA-PL) and MsbA proteonanodiscs (MsbA-ND); (ii) the ATPase activity of MsbA will be measured for MsbA-PL and MsbA-ND in the presence or absence of lipid A, LPS or drugs to clarify how different substrates affect the ATPase activity of MsbA; (iii) dynamics of MsbA on mica in the presence of various nucleotides will be imaged by HS-AFM. Indeed, these goals have been accomplished smoothly. Furthermore, "nanopatterned surfaces" for stably forming the freestanding lipid membranes incorporating MsbA transporters have been developed successfully for analyzing their conformational changes using HS-AFM. This assay system can be extended to study other ABC transporters.
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| Strategy for Future Research Activity |
To accomplish the final goals, the next crucial steps are (i) to analyze structure, dynamics and function of MsbA in lipid membranes bound different substrates (lipid A or LPS), nucleotides and drug inhibitors with sufficient spatio-temporal resolution; (ii) to compensate the limits in time and space resolutions of HS-AFM technique, it is important to combine with computational simulation for probing molecular structural dynamics of MsbA in action, with the quantitative comparison of simulation to AFM data and vice versa; (iii) to image simultaneously local and global structures of ABC transporters embedded in lipid membranes by the combined HS-AFM/single molecular FRET system; (iv) to study similarly with other transmembrane proteins.
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| Causes of Carryover |
In FY2020, I would like to continue our proposed experiments for accomplishing the final goals of this project. In practice, I would spend Kakenhi FY2020 budget for (i) personnel expenditure and remuneration; (ii) article cost; and (iii) travel expense. Based on the real demand in FY2020, I would combine and use all remaining budgets planned for personnel expenditure and remuneration (i.e., FY2020 + FY2021). This plan is to employ one to two technicians working for this project.
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