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Investigation of the pH dependent conformational changes of anthrax protective antigen and interactions with its cellular receptor
Rajapaksha, Maheshinie
Rajapaksha, Maheshinie
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Dissertation
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2011-05
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Electronic dissertations
Electronic dissertations
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Pathogenesis of anthrax is due primarily to the secretion of the anthrax toxin, a tripartite set of proteins that include protective antigen (PA), edema factor (EF), and lethal factor (LF). PA binds to the Von-Willebrand factor A (VWA) domain of a host cellular receptor and is then cleaved by a furin-like protease on the cell surface. Seven of such residues spontaneously oligomerizes to form a donut-shaped heptameric prepore. The prepore undergoes a major structural change at acidic pH that results in the formation of a membrane spanning pore, an event necessary for entry of EF and LF into the cell. Understanding the mechanism by which the protective antigen form a pore and translocate EF and LF could potentially guide the design of therapeutics aimed at preventing anthrax toxin entry.
The work presented in this thesis is an attempt to understand the pH dependent conformational changes of the prepore and the interactions of PA with its cellular receptor capillary morphogenesis protein2 (CMG2). Results presented in this thesis is divided into 3 parts. Chapter 3 summarizes the role of the receptor in the event of pore formation. We provide direct evidence using 1-dimensional 13C edited 1H NMR to show that the receptor dissociates from heptameric prepore at low pH. Chapter 4 summarizes the role of His residues in pH dependent pore formation and the temporal relationship between domain 2 and domain 4 during the structural transition form a prepore to a membrane spanning pore at low pH. In this section we provide evidence that domains 2 and 4 come closer in contact upon pore formation. Chapter 5 discusses the influence of pH on binding of PA to the von Willebrand factor A domain of CMG2. Fluorescence Resonance Energy Transfer (FRET) has been used extensively in this section to show that the affinity of PA to CMG2 increases at low pH.
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Dissertation(Ph.D.)--Wichita State University, College of Liberal Arts and Sciences, Dept. of Chemistry
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Wichita State University
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© Copyright 2011 by Kankani Thantri Maheshinie Rajapaksha. All rights reserved