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Show Protein Structure of the Bacterial Flagellar Motor Bacteria move by means of long helical filaments spun by motors in the cell membrane. The motors spin at speeds reaching 1,700 revolutions per second using energy from a membrane ion gradient. The molecular structure of the motor and the mechanism by which chemical energy is converted into mechanical energy are unknown. The goal of the research undertaken was to contribute to the ongoing effort to determine structures of motor parts. The flagellar motor has a rotating segment, or rotor, and a stationary segment, or stator. Three proteins are thought to be involved in rotation. FliG is on the rotor, whereas MotA and MotB form a complex that functions as the stator. MotA and MotB are membrane proteins, and are known to function in ion conduction for generation or torque. My research concerned the membrane embedded portions of the MotA/MotB complex. MotA has four membrane crossing segments and MotB has one. The spatial arrangement of these membrane segments was examined by using targeted disulfide crosslinking. When the amino acid cysteine is introduced in two positions in adjacent segments, they can form a disulfide bond when the two cysteine positions are in close proximity. By using a protein analysis method called polyacrylamide gel electrophoresis (PAGE), the cysteine-crosslinking can be detected. Bacterial cells containing the target proteins are dissolved in detergent, and the proteins are separated according to size by electrophoresis through gels. The crosslinked proteins are larger and thus migrate through the gel more slowly. By identifying pairs of positions where disulfide bonding occurs, the relative position of segments in the complex can be deduced. Data obtained from the crosslinking studies added support to a structural model in which each MotA/MotB complex forms two distinct ion channels. Due to the especially challenging nature of membrane protein structure studies, further development of these crosslinking methods may find general applicability in membrane protein biochemistry. Jeff Pettey Class Standing: Junior Major: Russian Faculty Sponsor: Associate Professor David Blair Biology Department E-mail: blair@bioscience.utah.edu |