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.
Date of Award
5-2013
Access Type
Campus Access
Document type
dissertation
Degree Name
Doctor of Philosophy (PhD)
Degree Program
Chemistry
First Advisor
Lynmarie K. Thompson
Second Advisor
Craig T. Martin
Third Advisor
Stephen J. Eyles
Subject Categories
Biochemistry | Chemistry
Abstract
The transmembrane signaling mechanism of bacterial chemotaxis receptors is thought to involve changes in receptor conformation and dynamics. The receptors function in ternary complexes with two other proteins, CheA and CheW, that form extended arrays in the membrane. Previous studies have shown that attractant binding induces a small piston displacement of one helix of the periplasmic and transmembrane domains towards the cytoplasm, but it is not clear how this signal propagates through the cytoplasmic domain to control the kinase activity of the CheA bound at the membrane-distal tip, nearly 200 Å away. The cytoplasmic domain has been shown to be highly dynamic, which raises the question of how a small piston could propagate through a dynamic domain to control CheA kinase activity. Furthermore, mutagenesis studies have led to proposals that signals propagate through this domain by generating antisymmetric changes in dynamics in different subdomains. A method for measuring dynamics of the receptor in functional complexes with CheA and CheW is needed to address these issues.
We have developed a novel method that combines vesicle template directed assembly with HDX-MS and we have demonstrated its utility by measuring global and local dynamics of the Asp receptor cytoplasmic fragment (CF) in functional, membrane-bound complexes with the signaling proteins CheA and CheW. This method extends HDX-MS to membrane-bound functional complexes that are not stable in detergent micelles, such as the chemoreceptor complex. The global dynamics data demonstrate that CF exhibits significantly slower exchange in functional complexes. This exchange is comparable to that of other proteins of similar structure and is more compatible with propagating signals from tiny conformational changes. The local dynamics data demonstrate changes in different subdomains with signaling state: the kinase-on state shows slower exchange in the methylation region and greater protection from exchange in the signaling region. This first direct measure of stabilization/destabilization of CF subdomains during signaling is being used to test and refine proposed models for signal propagation in the cytoplasmic domain of bacterial chemotaxis.
DOI
https://doi.org/10.7275/n8wd-f568
Recommended Citation
Koshy, Seena Smitha, "Differences Between Bacterial Chemotaxis Receptor Signaling States Revealed By a Novel Hydrogen Exchange Mass Spectrometry Approach" (2013). Doctoral Dissertations 1896 - February 2014. 468.
https://doi.org/10.7275/n8wd-f568
https://scholarworks.umass.edu/dissertations_1/468