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Show THE UNIVERSITY OF UTAH UNDERGRADUATE RESEARCH ABSTRACTS Spatial Organization of Flim and Flig in the Flagellar Motor of Escherichia Coli Using Site-Directed Crosslinking Akiko Saito (David F. Blair) Department of Biology University of Utah Many species of bacteria move by means of flagella turned by reversible rotary motor in the cell mem-brane.1 The rotational mechanism of a bacterial flagellum is of interest due to the application to pathogenic organisms. It is related to the Type III secretion system that infectious bacteria use to invade their hosts. These motors are built from about twenty-five distinct proteins and have a stationary part (stator) and a rotary part (rotor). Energy generation to drive rotation comes from either the proton gradient or (in some species) the sodium gradient across the plasma membrane. Among these proteins, five are the most essential for motor rotation. Two, called MotA and MotB, comprise the stator. The three others, FliG, FliM, and FliN, are directly involved in the rotation of the rotor. These rotor proteins form the "switch complex" in the flagellar basal body, which functions in flagellar assembly, rotation, and directional switching. The objective of our study is to examine the spatial organization of FliM and FliG in the switch complex in a bacterial flagellar motor. An overall image of the flagellar basal body has been obtained from electron microscopic imaging, and structures of the FliM and FliG proteins have been recently solved by X-ray crystallography. However, the details of how these two proteins are arranged are still in guestion. In order to probe FliG-FliM interaction, we have performed targeted cysteine cross-linking experiments. Specific residues on the surface of the proteins were mutated into cysteines with chemically reactive sulfhydryl groups. The mutations were made singularly and in pairs. Cross-linking of the proteins was induced with oxidizing reagents (copper phenanthroline or iodine) and products were then detected by polyacrylamide gel electrophoresis. The results indicate that FliM binds to the two different sites in FliG, which is consistent with the previous finding by Brown et al.2 Additional study will put strong constraints on the position and the orientation of FliM relative to FliG, providing a basis for a structural model of the switch complex. [1] Berg, Jeremy M., John L. Tymoczko, and Lubert Stryer. Biochemistry, 5th edition, Basingstoke: Houndmills, 2001. [2] Brown, Perry N., M. Terrazas, K. Paul, and D. F. Blair. 2007. Mutational analysis of the flagellar protein FliG: sites of interaction with FliM and implications for organization pfthe switch complex. J. Bacteriol. 189:305-312. •Qure 1. Sites of mutation in FliM and FliG. This model shows the crystal s tructure of FliM and FliG of thermotoga maritime labeled with the mutated ammo acid residue numbers. The numbers a re corresponding to those of escherichia coli. FliG is on the top and two subunits of FliM a re shown at the ottom. Red spheres indicate the sites of strong interaction between FliM and FliG. Green spheres indicate locations where no FliM and FliG interaction s observed. Orange spheres show intermediate interaction of FliM with FliG. The dotted lines indicate examples of FliG-FliM cross-linking sites. |