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. / "July 2006." / Includes bibliographic references (leaves 96-102).

Genetic code expansion allows the incorporation of non-canonical amino acids with a variety of new functional groups: fluorescent amino acids,1-3 azides,4-6 alkynes,5-10 and photocrosslinkers.4,11,12 This incorporation requires the evolution of new tRNA/aminoacyl tRNA sythetase pairs. Traditionally screenings of novel tRNA/aminoacyl tRNA synthetase pairs have been done in vivo. While these in vivo screenings have proven robust, they are limited in multiple ways: non-canonical amino acids (ncAAs) must be nontoxic and bioavailable. Furthermore, library size is limited by transformation efficiency. Lastly, in vivo screenings require substantial amounts of the target ncAA, which is often not available in large masses. In vitro screenings bypass these limitations: toxicity and bioavailibilty are no longer concerns. Library size can be expanded by several orders of magnitude as we are no longer limited by transformation efficiency. Lastly, because in vitro transcription/translation reactions are routinely conducted on the μL scale, ncAA usage can be minimized. We set out to use in vitro compartmentalization to further expand the code. In an in vitro compartmentalization screening, the water droplets in a water-in-oil emulsion serve as separate reaction chambers in which individual library members are transcribed and translated. Here we report optimization of S30 transcription/translation reactions. Optimizations include cell lysis method, reaction temperature, template amount, and T7 RNA polymerase amounts. Yields remained low and we transistioned into the use of PURExpress. Fluorohistidines are isosteric with histidine, but not isoelectronic.13 This change in environment results in a reduction of pKa. We set out to synthesize 4-fluorohistidine to use as a pH probe in several target proteins. A synthesis of 4-fluorohistidine was published in 1973.14,15 We were able to improve upon this synthesis by reducing cost and improving yield of a key step in the reaction. Next, small peptides with polyhistidine tags were translated in vitro using our 4-fluorohistidine. We are calling this polyhistidine tag incorporating 4-fluorohistidine our “hexafluorohistag.” Because of the reduced pKa of the 4-fluorohistidine, the hexafluorohistag showed affinity to Nickel-NTA resin even at reduced pH. This allowed for the purification of hexafluorohistagged peptides in the presence of traditional polyhistidine-tagged peptides.