Influence of 2 fluorohistidine on pore formation in the anthrax protective antigen
AdvisorBann, James G.
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Bacillus anthracis secretes a toxin which consists of three proteins that is the cause of the anthrax disease symptoms leading to death. They are called the protective antigen (PA), edema factor (EF) and lethal factor (LF). The three proteins self-assemble into toxic complexes after PA binding to its receptors present on host cells. The toxin receptor complexes are then internalized and acidic endosomal pH triggers pore formation by PA and translocation of the LF and/or EF into the cytosol. In this study, we labeled PA with 2-fluorohistidine, an analog of histidine with a dramatically reduced side-chain pKa, in order to test the hypothesis that histidine protonation triggers the pH dependent change from a prepore to a pore. We have analyzed its functional properties. It can be cut by furin or trypsin and it can also bind with one of its receptor, the VWA domain of CMG2 and form a heptamer as wild type PA. However, the pore formation can be blocked by this labeled protein when bound to CMG2. Independent experiments show that 2F-His labeled PA can also block translocation. By modifying the protein with 2F-His, we show that histidine in PA and CMG2 does not play as a pH-sensitive trigger in the pore formation process. We provide hypotheses for these findings.
Thesis (M.S.)--Wichita State University, College of Liberal Arts and Sciences, Dept. of Chemistry.
Includes bibliographic references (leaves 96-102).