Abstract:
The protein MreB is a bacterial actin homolog found in most non-spherical bacteria. It is known to function in cell shape determination and maintenance. MreB coordinates the peptidoglycan biosynthesis machinery to break the spherical symmetry and provide a specific shape to the cell. But recent studies on MreB from the bacterium Myxococcus xanthus revealed an inevitable role of MreB in gliding motility. In this thesis, we try to address the question that what makes Myxococcus xanthus MreB (MxMreB) different from other bacteria to carry out an additional function in gliding. With this in mind, this project focuses on the biochemical characterization of WT, ATPase mutant, and membrane binding mutants of MxMreB since MreB polymerization and membrane interactions are key to the gliding mechanism. To characterize the MreB, we purified the full-length wildtype (WT) protein. Along with the WT protein, the ATPase hydrolysis mutants and membrane binding mutants were also designed, cloned, and purified. All purified proteins were characterized for their ATPase activity. Our results found that the Gln141 residue at the active site of MxMreB does not affect ATP hydrolysis. The wt and membrane binding mutants behaved similarly in various liposome binding assays. As part of my thesis I also looked at the known structures of actin filaments and their interacting partners. By comparing the helical parameters of the actin filaments from
various organisms, we found that the filament structure across organisms is well conserved except for Plasmodium falciparum due to its less sequence similarity with mammalian actin. We also observed that cofilin-bound actin filaments deviate in helical parameters from the canonical value. Interface studies of actin and cofilin helped us connect to the deviation in filament structure with the actin filament severing by cofilin.
