Off-campus UMass Amherst users: To download campus access 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 talk to your librarian about requesting this dissertation through interlibrary loan.

Dissertations that have an embargo placed on them will not be available to anyone until the embargo expires.

Document Type

Open Access Dissertation

Degree Name

Doctor of Philosophy (PhD)

Degree Program

Chemistry

Year Degree Awarded

2018

Month Degree Awarded

February

First Advisor

Lynmarie K. Thompson

Subject Categories

Biochemistry, Biophysics, and Structural Biology

Abstract

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.

Share

COinS