<p>The cytoplasmic ring (C-ring) of the flagellar motor consists of three proteins: FliG, FliM, and FliN, each present in different copy numbers. These proteins perform the function of transmitting torque from the stators to the basal body, as well as regulating the rotational direction of the flagellum. Despite decades of study and great progress towards the understanding of the molecular details of the flagellum’s mode of action, substantial questions still remain about its detailed architecture and molecular mechanisms. Here we describe a series of in vitro and in vivo experiments designed to provide insight into the structure of the flagellar C-ring. </p><p> We begin this work by presenting a background of the forms of cellular motility and provide context for the flagellum within the great diversity of motility mechanisms. We then summarize the bacterial chemotaxis signal transduction system, one of the most deeply characterized signaling pathways within biology. Lastly we introduce the flagellum with a focus on C-ring structure and function. </p><p> The research portion begins with a characterization of the interaction between the FliG N-terminal domain (FliG<sub>N</sub>) and the C-terminal region of the flagellar membrane protein FliF (FliF<sub>C</sub>). We find that these two proteins interact strongly and that this interaction causes widespread conformational changes throughout FliG<sub>N</sub>. Based on NMR and other biophysical data we propose a binding site for FliF<sub>C</sub> centered on helix 1 of FliG<sub>N</sub>. </p><p> In the next section we further characterize the interaction between FliF<sub> C</sub> and FliG<sub>N</sub>. We generate a fusion FliF<sub>C</sub>-FliG<sub> N</sub> polypeptide and characterize this complex. Using spin labeling experiments we confirm our predicted interaction site between FliF<sub>C</sub> and FliG<sub> N</sub>. We also identify a novel interaction between an important hydrophobic patch on the FliG<sub>NM</sub> linker and FliG<sub>N</sub>. </p><p> Next we study and characterize the domain architecture of the full-length FliG<sub>NMC</sub> protein. By evaluating pair-wise domain interactions and comparing NMR spectra of numerous FliG constructs, in combination with in vivo experiments, we provide evidence that the FliG middle- (FliG<sub>M</sub>) and C-terminal (FliG<sub>C</sub>) domains interact in an intra-protomer manner. This model is in excellent accord with the 3D structure of the <i>Salmonella typhimurium</i> C-ring as derived from cryo-EM. </p><p> Lastly, we describe a number of experiments probing complex formation between FliG, FliM and FliN, with the aim of determining how these interactions are modulated by CheY binding. </p>
| Identifer | oai:union.ndltd.org:PROQUEST/oai:pqdtoai.proquest.com:3618776 |
| Date | 11 June 2014 |
| Creators | Levenson, Robert Herman |
| Publisher | University of California, Santa Barbara |
| Source Sets | ProQuest.com |
| Language | English |
| Detected Language | English |
| Type | thesis |
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