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dc.contributorWichita State University. Department of Chemistryen_US
dc.contributor.authorWimalasena, D. Shyamalien_US
dc.contributor.authorCramer, John C.en_US
dc.contributor.authorJanowiak, Blythe E.en_US
dc.contributor.authorJuris, Stephen J.en_US
dc.contributor.authorMelnyk, Roman A.en_US
dc.contributor.authorAnderson, David E.en_US
dc.contributor.authorKirk, Kenneth L.en_US
dc.contributor.authorCollier, R. Johnen_US
dc.contributor.authorBann, James G.en_US
dc.identifierAI22021/ U54 AI057160en_US
dc.identifier.citationBiochemistry. 2007 Dec 25; 46(51): 14928-36.en_US
dc.descriptionClick on the DOI link below to access the article (may not be free).en_US
dc.description.abstractThe action of anthrax toxin relies in part upon the ability of the protective antigen (PA) moiety to form a heptameric pore in the endosomal membrane, providing a portal for entry of the enzymic moieties of the toxin into the cytosol. Pore formation is dependent on a conformational change in the heptameric prepore that occurs in the neutral to mildly acidic pH range, and it has been hypothesized that protonation of one or more histidine residues triggers this transition. To test this hypothesis, we used biosynthetic methods to incorporate the unnatural amino acid analogue 2-fluorohistidine (2-FHis) into PA. 2-FHis is isosteric with histidine but resists protonation at physiological pH values due to a dramatically reduced side-chain pKa ( approximately 1). We found that 2-FHis-labeled PA was biologically inactive, as judged by its inability to deliver a model intracellular effector, LFN-DTA, to the cytosol of CHO-K1 cells. However, whereas 2-FHis blocked a conformational transition in the full-length PA83 protein in the pH 5-6 range, the pH dependence of prepore-to-pore conversion of (PA63)7 was unchanged from the wild-type protein, implying that this conversion is not dependent on His protonation. Consistent with this result, the labeled, trypsin-activated PA was able to permeabilize liposomes to K+ and retained pore-forming activity in planar phospholipid bilayers. The pores in planar bilayers were incapable, however, of translocating a model ligand in response to a transmembrane pH gradient or elevated voltage. The results indicate that protonation of residues other than His, presumably Glu and/or Asp side chains, triggers pore formation in vitro, but His residues are nonetheless important for PA functioning in vivo.en_US
dc.description.sponsorshipNIAID NIH HHS/ NIAID NIH HHSen_US
dc.publisherAmerican Chemical Societyen_US
dc.subjectResearch Support, N.I.H., Extramuralen_US
dc.subjectResearch Support, N.I.H., Intramuralen_US
dc.subjectResearch Support, Non-U.S. Gov'ten_US
dc.subject.meshAntigens, Bacterial/chemistryen_US
dc.subject.meshBacterial Toxins/chemistryen_US
dc.subject.meshCHO Cellsen_US
dc.subject.meshCell Membrane/metabolismen_US
dc.subject.meshCircular Dichroismen_US
dc.subject.meshElectrophoresis, Polyacrylamide Gelen_US
dc.subject.meshHistidine/analogs & derivativesen_US
dc.subject.meshHydrogen-Ion Concentrationen_US
dc.subject.meshModels, Molecularen_US
dc.subject.meshProtein Denaturationen_US
dc.subject.meshProtein Structure, Tertiaryen_US
dc.subject.meshProtein Transporten_US
dc.subject.meshAntigens, Bacterial/geneticsen_US
dc.subject.meshAntigens, Bacterial/metabolismen_US
dc.subject.meshBacterial Toxins/geneticsen_US
dc.subject.meshBacterial Toxins/metabolismen_US
dc.titleEffect of 2-fluorohistidine labeling of the anthrax protective antigen on stability, pore formation, and translocationen_US
dc.coverage.spacialUnited Statesen_US
dc.description.versionpeer revieweden_US
dc.rights.holderCopyright © 2007 American Chemical Societyen_US

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