The research presented here seeks to advance the understanding of the chemotaxis system in Escherichia coli through the use of a novel surface-templating approach. The chemosensory system in E. coli is an example of a two-component pathway and is common in many prokaryotes and is also found in eukaryotes. The system allows the cell to constantly sample the surrounding chemical environment, swimming toward attractants and away from repellants. The mechanism of signal propagation across the membrane to the kinase is not well understood. For many years a ligand-induced shift or rotation within a receptor dimer was thought to be alone responsible for signal propagation across the membrane bilayer. However, evidence for receptor clustering has mounted during the past decade from this lab and others. Inter-dimer interactions, more broadly, receptor clustering, has been observed making it possible for a signal to be propagated laterally as well as vertically through the membrane thus revealing a new avenue for communication and information processing within the cell. Receptor clustering is now believed responsible, in part or wholly, for the remarkable sensitivity, due to large gain, and also the system's ability to respond to a wide variety of environmental stimuli including pH, temperature, and many chemical signals. Reconstitution of this cluster, or 'brain' as referred to in some instances, is paramount in elucidating how this system functions and is the main goal of the research presented here. Reconstitution of membrane proteins has proved difficult, thus we have developed a new method to assemble a 2D array of activating cytoplasmic fragments (CFs) of the aspartate receptor of E. coli onto a lipid membrane. Histidine-tagged CFs of the Tar receptor were assembled on the surface of sonicated and extruded unilamellar vesicles via a lipid containing the nickel-nitrilotriacetic acid moiety as a headgroup. In the presence of the adaptor protein CheW, the protein kinase CheA bound to and was activated up to 475-fold by vesicle-bound CF. Surface-assembled CF was also found to serve as a substrate of the receptor methyltransferase, CheR. Since neither significant CheA activation nor CF methylation was observed in comparable samples in the absence of vesicles, it was concluded that surface-templating generates the organization among CF subunits required for biochemical activity. This novel reconstitution method will be generally applicable to other systems where signaling occurs at or near the membrane surface and also for the generation of new anchoring chemistries for the marriage of proteins to surfaces.
| Identifer | oai:union.ndltd.org:UMASS/oai:scholarworks.umass.edu:dissertations-4347 |
| Date | 01 January 2006 |
| Creators | Shrout, Anthony L |
| Publisher | ScholarWorks@UMass Amherst |
| Source Sets | University of Massachusetts, Amherst |
| Language | English |
| Detected Language | English |
| Type | text |
| Source | Doctoral Dissertations Available from Proquest |
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