Structure Biology of Rotational Catalysis in F1-ATPase

F1-ATP酶旋转催化的结构生物学

基本信息

批准号：
10480186
负责人：
SHIRAKIHARA Yasuo
金额：
$ 7.23万
依托单位：
National Institute of Genetics
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B).
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-10480186/
关键词：
F1-ATPASE ATPsynthase Cryslallization α3β3-complex α3β3γ-complex α3β3γε-complex Crystal structure F1ATPase F_1ATPase

项目摘要

F1-ATPase, with a subunit composition of α3β3γδε, is a catalytic sector of the membrane bound ATP synthase. The rotational catalysis mechanism of F1 is accompanied by rotation of the rod-like g subunit. We previously solved the structure of the a3b3 sub-assembly of F1-ATPase from a thermophilic bacterium Bacillus PS3. We have extended the structural study to nuceotide-bound a3b3 subassembly and α3β3γ and α3β3γε sub-assemblies.We looked at the structure of the nucleotide-bound form of the α3β3 sub-assembly by analyzing x-ray diffraction data from the nucleotide-soaked crystals. We could trap a state where the over-all structure is very similar to that of the nucleotide-free state, but where phosphate binding to the β-subunit gets weaker than in the nucleotide-free state. Also we have tried to crystallize the sub-assembly in presence of non-hydrolyzable nucleotide analog AMPPNP, and got crystals.We have made considerable efforts to get good crystals of the α3β3γ sub-assembly. Both preparation method and crystallization conditions have been examined extensively. We found that those crystals diffracted to resolutions of 15-10A at SPring8. Although the values were poor, they were twice better than values for resolution limit obtained with laboratory x-ray source. Further efforts are being made to extend the resolution of the crystals and to find appropriate conditions for cooling crystals.The α3β3γε sub-assembly crystals have been found to be much better than the α3β3γ sub-assembly crystals described above. With SPring8 beam, we collected a data set to resolution of 4.5 A at 100K.The unit cell parameters were a=225.3A, b=225.7A, c=224.6A, α=94.1, β=117.5, γ=117.8, and the unit cell contains 8 of the sub-assembly. The data were merged with Rmerge of 12.2% at 4.5 A resolution. Structural analysis by molecular replacement is in progress.

F1-ATPase 的亚基组成为 α3β3γδε，是膜结合 ATP 合酶的催化部分，F1 的旋转催化机制伴随着杆状 g 亚基的旋转。从嗜热细菌芽孢杆菌 PS3 组装 F1-ATPase 我们已将结构研究扩展到核苷酸结合的 a3b3。我们通过分析来自核苷酸浸泡晶体的 X 射线衍射数据来研究 α3β3 子组件的核苷酸结合形式的结构，我们可以捕获整体的状态。结构与无核苷酸状态非常相似，但磷酸盐与 β-亚基的结合比无核苷酸状态弱。在不可水解的核苷酸类似物AMPPNP存在下使该子组装体结晶，并得到晶体。我们为获得良好的α3β3γ子组装体晶体进行了广泛的研究，我们发现这些方法和结晶条件。晶体在 SPring8 上的衍射分辨率为 15-10A，尽管这些值很差，但比实验室 X 射线源获得的分辨率极限值好两倍。已发现 α3β3γε 子组件晶体比上述 α3β3γ 子组件晶体要好得多，我们使用 SPring8 光束收集了数据集。 100K 时分辨率为 4.5 A。晶胞参数为 a=225.3A、b=225.7A、c=224.6A， α=94.1，β=117.5，γ=117.8，并且晶胞包含 8 个子组件。数据以 4.5 A 分辨率进行 12.2% 的合并。正在进行分子替换的结构分析。