Off-campus UMass Amherst users: To download dissertations, please use the following link to log into our proxy server with your UMass Amherst user name and password.
Non-UMass Amherst users, please click the view more button below to purchase a copy of this dissertation from Proquest.
(Some titles may also be available free of charge in our Open Access Dissertation Collection, so please check there first.)
Receptor clusters and dynamics in kinase regulation in bacterial chemotaxis
Signal transduction in bacterial chemotaxis system is mediated by a family of transmembrane proteins that are clustered in the membrane and form noncovalent complexes with cytoplasmic signaling proteins. Recent evidence suggests that receptor clusters play an important role in mediating the signaling in bacterial chemotaxis. To understand the functional role of receptor clusters in kinase regulation and the mechanism of cluster formation, we employed a strategy to engineer an intrasubunit disulfide bond between the two methylation regions in the cytoplasmic domain of E. coli aspartate receptor (Tar CF). We assumed the potential stabilizing effect of an internal disulfide bond might change receptor's propensity to cluster. Furthermore, the intrasubunit disulfide bond effectively prevents domain swapping, thus providing a tool to test the recently proposed "domain swapping model" as a mechanism that generates cluster formation and CheA activation. Our results showed one disulfide-bonded CF, termed CFSP3, exhibited a slight increase in protein stability together with a dramatic increase in CheA activation, high cooperativity and a strong propensity to form large receptor clusters in solution. Conversely, we did not observe notable cluster formation in any CFs in solution that failed to activate CheA. Yet when these assembled into clusters by templating methods, some measure of CheA activation was achieved. We also found that addition of CheW significantly decreased the cluster size, suggesting CheW might bind to the same region as where Us make contact to oligomerize. The results demonstrate that domain swapping is not required for the formation of active receptor clusters. We also propose a clustering model in which a receptor's propensity to form large clusters correlate with the receptor-coupled kinase activity in solution. Tar CF is highly dynamic, and the regulation of dynamics has been proposed to be involved in signaling. Therefore, HDX properties of the engineered CFs were studied to gauge dynamics. Overall Us showed near complete exchange, although a detectable difference in HDX protection was observed between the reduced and oxidized forms of CFSP3, which may be functionally significant. Future experiments conducted with ternary complexes should provide invaluable information to clarify the functional role of receptor dynamics in signaling.
Zhi, Li, "Receptor clusters and dynamics in kinase regulation in bacterial chemotaxis" (2005). Doctoral Dissertations Available from Proquest. AAI3193963.