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Date of Award
Doctor of Philosophy (PhD)
Lynmarie K. Thompson
Craig T. Martin
Richard W. Vachet
Biochemistry | Microbiology
Both clusters and conformational changes are thought to be important in the transmembrane signaling mechanism of bacterial chemotaxis receptors. Full signaling activity of these receptors requires assembly of a ternary complex with two other proteins, CheA and CheW, and these complexes form hexagonal arrays that localize at the poles of the cell. The functional role of clustering of chemoreceptors is a controversial issue in chemotaxis signaling. A few research groups have evidence suggesting that attractant binding can alter receptor clustering, whereas other evidence shows only minor effects of attractant. In order to test whether ligand binding modulates a clustering equilibrium to control the kinase activity of CheA, we have reconstituted the intact receptor into membrane vesicles at a range of lipid-to-receptor ratios comparable to the physiological range in bacteria. Activity measurements demonstrate that the intact, unmethylated receptor activates CheA to a similar extent over this receptor concentration range. Ligand affinity of receptors in complexes, measured by inhibition of kinase activity, is also independent of concentration for the unmethylated receptor, indicating that ligand binding does not alter receptor clustering. A construct that mimics the fully methylated form of the reconstituted receptor also exhibits concentration-independent kinase activation. These results suggest that receptor clustering does not play a role in the primary signaling event, control of kinase activity.
Consolacion, Fe C, "Clustering Independence Of Ligand Affinity And Kinase Activity Of Reconstituted Bacterial Chemoreceptors: Insight Into Signaling Mechanisms" (2011). Doctoral Dissertations 1896 - February 2014. 266.