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Date of Award
Doctor of Philosophy (PhD)
Lynmarie K. Thompson
Craig T. Martin
Stephen J. Eyles
Biochemistry | Chemistry
The transmembrane signaling mechanism of bacterial chemotaxis receptors is thought to involve changes in receptor conformation and dynamics. The receptors function in ternary complexes with two other proteins, CheA and CheW, that form extended arrays in the membrane. Previous studies have shown that attractant binding induces a small piston displacement of one helix of the periplasmic and transmembrane domains towards the cytoplasm, but it is not clear how this signal propagates through the cytoplasmic domain to control the kinase activity of the CheA bound at the membrane-distal tip, nearly 200 Å away. The cytoplasmic domain has been shown to be highly dynamic, which raises the question of how a small piston could propagate through a dynamic domain to control CheA kinase activity. Furthermore, mutagenesis studies have led to proposals that signals propagate through this domain by generating antisymmetric changes in dynamics in different subdomains. A method for measuring dynamics of the receptor in functional complexes with CheA and CheW is needed to address these issues.
We have developed a novel method that combines vesicle template directed assembly with HDX-MS and we have demonstrated its utility by measuring global and local dynamics of the Asp receptor cytoplasmic fragment (CF) in functional, membrane-bound complexes with the signaling proteins CheA and CheW. This method extends HDX-MS to membrane-bound functional complexes that are not stable in detergent micelles, such as the chemoreceptor complex. The global dynamics data demonstrate that CF exhibits significantly slower exchange in functional complexes. This exchange is comparable to that of other proteins of similar structure and is more compatible with propagating signals from tiny conformational changes. The local dynamics data demonstrate changes in different subdomains with signaling state: the kinase-on state shows slower exchange in the methylation region and greater protection from exchange in the signaling region. This first direct measure of stabilization/destabilization of CF subdomains during signaling is being used to test and refine proposed models for signal propagation in the cytoplasmic domain of bacterial chemotaxis.
Koshy, Seena Smitha, "Differences Between Bacterial Chemotaxis Receptor Signaling States Revealed By a Novel Hydrogen Exchange Mass Spectrometry Approach" (2013). Doctoral Dissertations 1896 - February 2014. 468.