The MotA and MotB proteins of Escherichia coli together form a proton- conducting membrane channel that is essential for flagellar rotation. Homologs of the MotA/B channel exist in many species of motile bacteria, including pathogens whose virulence is linked with motility. The structure of this channel, and the relation of its structure to its ion-conducting function, are being studied using molecular genetic and biochemical approaches. These studies have resulted in a hypothesis for the gross structural features of the channel, and suggest that the proton conduction pathway must consist largely of water molecules contained in the channel. Targeted chemical crosslinking and other biochemical approaches will be used to refine and extend this model. Based on these studies, it will be possible to formulate a detailed hypothesis for the MotA/B channel structure and its mechanism of proton conduction. This will assist in understanding other processes where proton movement is essential, including the energy conversions that occur in mitochondria, chloroplasts, and bacteria. The MotA/B channel functions as part of the flagellar motor, a large complex containing about twenty proteins. The parts of MotA/B that extend into the cytoplasm of the cell are believed to participate in torque generation by the motor. In order to identify the parts that are most closely involved in torque generation, the structure-function study will be extended to the cytoplasmic parts of both proteins, using intensive random mutagenesis coupled with detailed assays of function. The cytoplasmic parts of MotA will also be expressed separately from its hydrophobic, membrane-spanning parts and purified in preparation for structural studies. Additionally, photoactivated crosslinking studies will be carried out to identify and approximately locate proteins close to MotA/B in the flagellar motor. This will provide insight into the arrangement of the components that generate torque. The objective of these studies is to understand, at the molecular level, the mechanism of torque generation in the flagellar motor.
