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Master of Science (M.S.)
Year Degree Awarded
Month Degree Awarded
Vesicle assembled approach to determine the protein oligomer behavior by site directed cross-linking
Chemoreceptor dimers mediate bacterial chemotaxis through receptor arrays that form complexes with CheW and the kinase CheA. Both kinase activity and the rate of receptor methylation have been observed to change with receptor density in the signaling complexes of an assembled system. Receptor dimers play a structural and functional important role during the signaling process. The dimer is an established unit of a functional receptor array, but the exact packing arrangement of dimers in the signaling array is not known in detail. Two different models of the packing arrangement have been proposed, based on crystal structures of receptor cytoplasmic fragments: the trimer of dimers and hedgerow models. Here, we determine the importance of CF dimer organization on density dependence of kinase activity, receptor methylation, and we probe CF trimer organization by site-directed TMEA cross-linking. CF dimers were prepared by site-directed cysteine disulfide bond formation to test for a possible monomer-dimer equilibrium process in the density-dependence of kinase activity in the vesicle-assembled system. We found that density transition observed for CF4E is similar for both reduced and disulfide-linked CF, indicating that the CF is organized as dimers even at lower densities. CheR-catalyzed methylation experiment revealed that disulfide-linked CF dimers in solution are insufficient for efficient methylation, assembly of the CFs on a vesicle surface is required. CF trimers were trapped by the trifunctional crosslinker TMEA, but only with vesicle assembled CFs. TMEA crosslinking occurred between residues that were expected to form trimer, but also those that were not. The results provide evidence that CF behavior is highly dynamic in maleimide crosslinking reaction which conducted on vesicle assembled system.
Robert M Weis