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

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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