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. ***
