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Study on structure and function of force-generating proteins in ion-driven flagellar motor

离子驱动鞭毛运动中力产生蛋白的结构和功能研究

基本信息

批准号：
13308038
负责人：
HOMMA Michio
金额：
$ 33.7万
依托单位：
Nagoya University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13308038/
关键词：
ion channel energy transduction Vibrio flagella motor Sodium motive force 細菌べん毛運動静電相互作用

项目摘要

The bacterial flagellar motor is the molecule machine which changes ionic inflow into the rotational power, and it is divided into the Na^+-and H^+-driven type by the coupling ions. Four components of PomA, PomB, MotX and MotY are necessary for the polar flagella driven by Na^+ in the oceano bacteria, Vibrio alginolyticus. We made chimera proteins between the H^+-driven type MotAB of E.coli and the Na^+-driven type PomAB of Vibrio alginolyticus, and die functions of these chimera proteins were analyzed. MotXY are not necessary for the function of the chimera motor that consists of the extracelluler region of the H^+-driven type MotB protein and the transmembrane region of the Na^+-driven type PomB protein. This chimera motor is functional even in E.coli cells as Na^+-driven type. Furthermore, we could isolate the temperature sensitivity mutant whose mutation site is determined in the motor proteins. This kind of mutant had not been isolated. From the analysis about this temperature sen … More sitivity, it was clarified that the neighborhood area of the transmembrane regions (TM2 and TM3) connected to the cytoplasmic region of PomA is important for the motor function. Using the PomA and PomB proteins fused to GFP, we investigated the assemble process of the stator proteins which is essential for the energy transduction. We could observe the fluorescent dots at the cell poles accompanied with the flagellar structure. The stator proteins formed a complex in the inner membrane. From the results using this GFP experimental system, we could propose the model that the motor proteins are incorporated as the complex into the flagellar motor. Finally, we found the motor protein, MotY, was localized in the periplasmic space without MotX and its overexpression system was constructed. The large amount of MotY could be prepared to examine the conditions for the crystal formation of MotY. As the result, we could have the crystals with hexagonal pillar, and get the diffraction pattern with the 2.7Å resolution. We are going to determine its phase and to make the molecular modeling of the MotY structure. Less

细菌鞭毛马达是将离子流入转化为旋转动力的分子机器，根据耦合离子的不同分为Na^+驱动型和H^+驱动型。PomA、PomB、MotX和MotY是奥恰诺中Na^+驱动极鞭毛所必需的四种组分。我们将大肠杆菌的H^+驱动型MotAB和溶藻弧菌的Na^+驱动型PomAB构建成嵌合蛋白，并对这些嵌合蛋白的功能进行了分析。嵌合体马达由H^+驱动型MotB蛋白的细胞外区和Na^+驱动型PomB蛋白的跨膜区组成，MotXY对于嵌合体马达的功能不是必需的。这种嵌合体马达甚至在大肠杆菌细胞中也是Na^+驱动型的。此外，我们可以分离的温度敏感性突变体，其突变位点确定在马达蛋白。这种变异体还没有被分离出来。通过对该温度传感器的分析， ...更多信息通过对PomA的跨膜区（TM2和TM3）的活性的研究，阐明了与PomA的胞质区连接的跨膜区（TM2和TM3）的邻近区域对于运动功能是重要的。利用与GFP融合的PomA和PomB蛋白，我们研究了能量转导所必需的定子蛋白的组装过程。在细胞两极可观察到荧光点，并伴有鞭毛结构。定子蛋白在内膜中形成复合物。从使用该GFP实验系统的结果，我们可以提出马达蛋白作为复合物并入鞭毛马达的模型。最后，我们发现马达蛋白MotY定位于周质间隙，而MotX不存在，并构建了MotY的过表达系统。可以制备大量的MotY以检查MotY晶体形成的条件。实验结果表明，我们可以得到六方柱晶体，并得到分辨率为2.7 μ m的衍射图样。我们将确定它的相位，并对MotY结构进行分子建模。少