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具有α3β3γδε的亚基组成，是膜结合ATP合酶的催化扇区。 F1的旋转催化机制是通过杆状G亚基的旋转来完成的。我们先前从嗜热细菌PS3中求解了F1-ATPase的A3B3子组装的结构。我们已经将结构研究扩展到结合了甲状腺素结合的A3B3亚组装，以及α3β3γ和α3β3γε子组件。我们通过分析的X-ray衍射数据从核苷酸浸出的数据中研究了α3β3子组装的核苷酸结合形式的结构。我们可以捕获一个与无核苷酸状态非常相似的状态，但是与无核苷酸状态相比，磷酸盐与β-亚基的结合要弱。同样，我们也试图在存在非氢化核苷酸模拟AMPPNP的情况下将子组装结晶，并获得了晶体。我们已经做出了考虑的努力，以获得良好的晶体良好的晶体。制备方法和结晶条件均已广泛研究。我们发现，这些晶体在弹簧8时衍射为15-10a的分辨率。尽管值很差，但它们的分辨率极限的值是实验室X射线源获得的分辨率极限的两倍。正在采取进一步的努力来扩展晶体的分辨率并找到适当的冷却晶体条件。已经发现α3β3γε子组装晶体比上述α3β3γ亚组装晶体要好得多。使用Spring8 Beam，我们收集了一个数据集以100K的分辨率分辨率为4.5A。单位细胞参数为A = 225.3a，B = 225.7a，C = 224.6a，α= 94.1，β= 117.5，γ= 117.8，并且单位电池包含子组件的8个。在4.5 A分辨率下，数据与12.2％的rmerge合并。通过分子置换进行结构分析正在进行中。