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Structural elucidation of active ion transport

活性离子传输的结构阐明

基本信息

批准号：
10044198
负责人：
TOYOSHIMA Chikashi
金额：
$ 3.01万
依托单位：
The University of Tokyo
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 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10044198/
关键词：
active transport protein crystal membrane proteins ion pump over expression electron crystallography X-ray crystallography ATPase

项目摘要

The aim of this project is to elucidate the mechanism of active transport from the structural standpoint. When this project was proposed, we had merely two kinds of crystals of CaィイD12+ィエD1-ATPase useful for electron microscopy. Since then, we succeeded in generating large crystals suitable for X-ray crystallography and expected to solve the structure at an atomic resolution within two years or so. Therefore, we shifted the stress on considering on the utilization of mutants for structural studies and asked Prof. Inesi of the University of Maryland to join, because his group was the only one that-succeeded in making large quantity of this membrane protein. This collaboration went out extremely well and produced one review and one original research paper (submitted to Biochemistry). The mutational studies agreed very well with the structure revealed by x-ray crystallography (submitted to Nature) and provided insights into the conformational changes required for active transport. On the other hand, the structure was indispensable to interpret the biochemical and mutational results correctly. Altogether, this project became a very good example of international collaboration. The next step is obviously to make crystals of mutants. For this purpose, the purification procedure used for SR CaィイD12+ィエD1-ATPase has turned out to be insufficient. We are now improving the methods for cultured cells in collaboration with Prof. Inesi.

本项目的目的是从结构的角度阐明主动转运的机制。当本项目提出时，我们只有两种用于电子显微镜的CaィイD12+ィエD1-ATPase晶体。从那时起，我们成功地产生了适合于X射线结晶学的大晶体，并有望在两年左右的时间内以原子分辨率解决结构问题。因此，我们将重点转移到考虑利用突变体进行结构研究，并邀请了马里兰大学的伊内西教授加入，因为他的团队是唯一一个成功地大量制造这种膜蛋白的团队。这次合作进行得非常顺利，产生了一篇综述和一篇原创研究论文(提交给生物化学)。突变研究与X射线结晶学(提交给《自然》)揭示的结构非常吻合，并提供了对主动运输所需的构象变化的见解。另一方面，结构对于正确解释生化和突变结果是必不可少的。总而言之，这个项目成为国际合作的一个很好的例子。下一步显然是制造变种人的晶体。为此，目前对SR CaィイD12+ィエD1-ATPase的纯化方法还不够完善。我们现在正在与伊内西教授合作改进培养细胞的方法。