| 研究課題/領域番号 |
17H03647
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| 研究機関 | 大阪大学 |
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研究代表者 |
Gerle Christoph 大阪大学, たんぱく質研究所, 特任准教授(常勤) (10561970)
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| 研究期間 (年度) |
2017-04-01 – 2020-03-31
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| キーワード | FoF1 ATP synthase / mitochondria / bioenergetics / single particle cryo-EM / structural biology / membrane biology / proton transport / rotary ATPase |
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| 研究実績の概要 |
For advancing our understanding of mammalian FoF1 ATP synthase structure and function at the atomic level via structural analysis by single particle cryo-EM the following experimental results were achieved during the first year of this Kiban B project:
(1) To circumvent the destabilization of the complex by contact with column resing through the loss of native bound lipids such as cardiolipin we are establishing a column free purification procedure. Initial success has been found and progress looks promising for future improvements of conditions recently found.
(2) We have succeeded to solubilize large amounts of the mammalian FoF1 ATP synthase without compromising the complexes’ stability by optimizing the composition and amount of novel detergents.
(3) Testing of many differenct freezing conditions allowed us find cryo-grid specimen preparation approaches that do not destruct the highly fragile mammalian FoF1 ATP synthase.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
Challenges in large scale purifications are becoming solvable, which is also true for technical issues in image data acquisition.
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| 今後の研究の推進方策 |
Conformational flexibility is a major obstacle for structural characterization by any method and this is also true for single particle cryo-EM. To mitigate this in the case of rotary ATP synthases serious problem we will aim to establish purification procedures that keep physiological bound IF1 bound to the complex during the isolation procedure.
The fact that mitochondrial FoF1 ATP synthase exists in oligomeric form in the inner mitochondrial membrane complicates structural analysis. We will establish efficient protocols for the large separation of the different oligomeric forms and analyze them separately.
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