Mechanism of Flagellar Rotation in Escherichia coli

9513486布莱尔许多种类的细菌是由可逆的旋转马达推动的，这些马达从穿过细胞膜的质子梯度中获得旋转的能量。因此，旋转马达是一种将化学能转换为机械能的换能器，效率非常高。在组装和操作这些马达所需的许多蛋白质中，只有几种蛋白质被认为直接在扭矩产生中发挥作用。其中包括MOTA和MOTB，它们形成了穿过细胞膜的质子通道，以及FliG、Flim和Flin，它们的确切功能尚不清楚。最近的工作表明，FliG、FliM和FLIN形成了一个可以在体外检测到的复合体，FliG广泛地参与了扭矩的产生过程，而FliM和FLIN则不参与。此外，在FliG的C末端有一个相对较小的结构域被证明在扭矩产生中具有特定和广泛的功能。在了解细菌鞭毛旋转机制方面的持续进展将要求使用接头扫描突变结合现有的结合分析来绘制FliG、Flim和Flin蛋白之间的相互作用图。FliG C-末端结构域将进行密集的突变研究，并将进行纯化和鉴定，为结构研究做准备。这些蛋白质在鞭毛中的位置仅大致为人所知。为了更准确地定位蛋白质和某些感兴趣的蛋白质结构域，将使用分子遗传学方法来低表达蛋白质，表达蛋白质的亚结构域，或者表达可以用电子致密探针特异性标记的形式，并在电子显微镜中检查由此产生的鞭毛结构。这些研究的最终目标是在分子水平上了解鞭毛马达如何将化学能转化为机械能。拟议的研究将通过澄清关键蛋白质成分的安排和功能，在实现这一目标方面取得重大进展。了解能量转换的机制，特别是那些涉及质子运动的机制，是生物能量学的核心问题。此外，生物分子机器的这个例子可能是纳米级马达或开关的纳米制造工程的有用模型。***

9513486 Blair Many species of bacteria are propelled by reversible rotary motors that derive the energy for rotation from the proton gradient across the cell membrane. The rotary motor is thus a transducer that converts chemical energy into mechanical energy, with very high efficiency. Among the many proteins needed to assemble and operate these motors, only a few have been suggest to function directly in torque generation. These include MotA and MotB, which form a proton channel across the cell membrane, and FliG, FliM and FliN, whose precise functions are not known. Recent work has shown that FliG, FliM, and FliN form a complex that can be detected in vitro, and that FliG is extensively involved in the process of torque generation whereas FliM and FliN are not. Further, a relatively small domain at the C-terminus of FliG was shown to function specifically and extensively in torque generation. Continued progress in understanding the mechanism of flagellar rotation in bacteria will require that the interactions among the FliG, FliM, and FliN proteins will be mapped using linker-scanning mutagenesis coupled with existing binding assays. The FliG C-terminal domain will be characterized by intensive mutational studies, and it will be purified and characterized in preparation for structural studies. The locations of these proteins in the flagellum is known only approximately. To locate the proteins and certain protein domains of interest more precisely, molecular genetic approaches will be used to underexpress the proteins, express subdomains of the proteins, or express forms that can be specifically labeled with an electron-dense probe, and the resulting flagellar structures will be examined in the electron microscope. The ultimate goal of these studies is to understand, at the molecular level, how the flagellar motor converts chemical energy into mechanical energy. The proposed studies will achieve significant progress toward that goal by clarifying the arrangement and function of the key protein components. Understanding mechanisms of energy conversion, particularly those that involve proton movements, is a central concern in bioenergetics. Also, this example of a biomolecular machine may be a useful model for nanofabrication engineering of nanoscale motors or switches. ***