Structure-Function Relationships in the Spirochetal Flagellar Motor

描述(申请人提供)：细菌的运动性及其驱动力，鞭毛马达，是伯氏杆菌(莱姆病的病原体)和许多其他细菌的重要毒力因素。鞭毛马达是一种非凡的纳米机器，由穿过细胞质膜的质子(或钠)梯度提供动力。电化学梯度与机械旋转的耦合是这台分子机器最吸引人的特征之一。鞭毛电机由两个主要部件组成：定子和转子。尽管先前的结构研究揭示了鞭毛转子的惊人复杂性，但鞭毛电机中的能量耦合机制在分子水平上仍然知之甚少，这主要是因为缺乏关于鞭毛旋转所涉及的膜结合定子和转子-定子相互作用的结构信息。中心假设是，转子-定子-C环界面的高分辨率结构将提供提出旋转和反转机制模型所需的重要结构信息。本应用的目的是通过将新型高通量冷冻电子断层扫描(Cryo-Et)方法与遗传分析相结合，原位确定完整鞭毛马达的结构/功能关系，以研究模型系统伯氏杆菌。通过与史蒂文·诺里斯、尼尔斯·查隆博士合作。Md Motaleb，Chunho Li和Hanspeter Winkler，我们建议专注于两个特定的目标：特定目标1-通过分析2 nm分辨率的完整鞭毛电机和纯化的鞭毛转子的三维结构来确定扭矩产生单元的详细结构。具体目标2-通过对野生型生物和鞭毛基因突变的比较分析，确定单个鞭毛蛋白的结构和功能作用。我们认为，对转子/定子组件的现场详细分析可能为理解鞭毛旋转和细菌运动的机制提供最清晰的途径，这反过来将适用于所有螺旋体和其他运动细菌的发病机制。此外，作为该项目的一部分，高通量Cryo-ET的进一步开发将容易地应用于获得与各种人类病原体的发病机制相关的大分子机器的结构和功能关系的新见解，并在活的生物体中提供广泛的分子分辨率的重要生物医学信息。

DESCRIPTION (provided by applicant): Bacterial motility and its driving force, the flagellar motor, are important virulence factors of B. burgdorferi (the causative agent of Lyme disease) and many other bacteria. The flagellar motor is a remarkable nano-machine, powered by the proton (or sodium) gradient across the cytoplasmic membrane. The coupling of an electrochemical gradient to mechanical rotation is one of the most fascinating features of this molecular machine. The flagellar motor is composed of two major components: the stator and the rotor. Although prior structural studies have revealed the stunning complexity of the flagellar rotor, the mechanism of energy coupling in the flagellar motor remains poorly understood at the molecular level, mainly because of the lack of structural information about the membrane-bound stator and the rotor- stator interactions involved in flagellar rotation. The central hypothesis is that a high- resolution structure of the rotor-stator-C ring interface will provide vital structural information needed to propose models on the mechanisms of rotation and reversal. The objective of this application is to determine the structure/function relationship of the intact flagellar motor in situ by combining novel high throughput Cryo-Electron Tomography (Cryo-ET) approaches with genetic analysis to study the model system, B. burgdorferi. By collaborating with Drs. Steven Norris, Nyles Charon. MD Motaleb, Chunhao Li and Hanspeter Winkler, we propose to focus on two specific aims: Specific Aim 1 - Determine the detailed structure of the torque-generating unit by analyzing the 3-D structures of the complete flagellar motor and the purified flagellar rotor at 2 nm resolution. Specific Aim 2 - Determine the structural and functional roles of individual flagellar proteins by comparative analysis of wild-type organisms and flagellar gene mutants. We believe that the detailed analysis of the rotor/stator assembly in situ may provide the clearest avenue yet available to understanding of the mechanism of flagellar rotation and bacterial motility, which will in turn be applicable to the pathogenesis of all spirochetes and other motile bacteria. In addition, the further development of high-throughput Cryo-ET as part of this project will be readily applied to gain new insights into the structural and functional relationship of macromolecular machines related to pathogenesis of a variety of human pathogens, and offer a wide spectrum of important biomedical information at molecular resolution in living organisms.