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Author ORCID Identifier
Open Access Dissertation
Doctor of Philosophy (PhD)
Year Degree Awarded
Month Degree Awarded
Lynmarie K. Thompson
Biochemistry, Biophysics, and Structural Biology
Transmembrane chemotaxis receptors are found in bacteria in extended hexagonal arrays stabilized by the membrane and by cytosolic binding partners, the kinase CheA and coupling protein CheW. Models of array architecture and assembly propose receptors cluster into trimers-of-dimers that associate with one CheA dimer and two CheW monomers to form the minimal "core unit" necessary for signal transduction. Reconstructing in vitro chemoreceptor ternary complexes that are homogenous, functional, and exhibit native architecture remains a challenge. Here we report that His-tag mediated receptor dimerization with divalent metals is sufficient to drive assembly of native-like functional arrays of a receptor cytoplasmic fragment. Our results indicate receptor dimerization initiates assembly and precedes formation of ternary complexes with partial kinase activity. Restoration of maximal kinase activity coincides with a shift to larger complexes, suggesting that kinase activity depends on interactions beyond the core unit. We hypothesize that achieving maximal activity requires building core units into hexagons and/or coalescing hexagons into the extended lattice. This discovery may also address a previously observed density-dependent transition between signaling states. To further test this, we implemented a paramagnetic relaxation enhancement (PRE) based solid-state NMR approach to obtain long-range (≥ 20 Å) distance constraints across the trimer of dimers interface. Overall, the work presented here shows that minimally perturbing His-tag mediated dimerization promotes assembly of chemoreceptor arrays with native architecture, and thus enabled us to gain insights into the mode of array assembly and the role of the core functional unit.
Haglin, Elizabeth R., "Assembly and Functional Architecture of Bacterial Chemoreceptor Nanoarrays" (2018). Doctoral Dissertations. 1171.