The anthrax toxin channel: Ion conductance, ion selectivity, and protein translocation
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The tripartite toxin produced by Bacillus anthracis is the causative agent of anthrax. The toxin consists of three protein components: lethal factor (LF), edema factor (EF), and protective antigen (PA). The C-terminal, 63 kD portion of PA heptamerizes to form channels in cell membranes that allow the passage of LF and EF into the cytosol, where these two enzymes exert their toxic effects. The channels are cation-selective and mushroom-shaped, with a globular cap domain and a cylindrical stem domain. When the PA channels are reconstituted in planar phospholipid bilayers, EF and LF can be driven through the channels by applying either a DeltapH or a DeltaPsi across the membrane. In this thesis, I first examined ion conductance in the stem of the anthrax toxin channel. While it is known that the Ф-clamp (a ring of phenylalanine residues located at the junction of the channel cap and stem domains) is responsible for causing an essentially complete block in conductance during protein translocation, it is unclear whether any ions may pass through the channel stem when occupied by a protein substrate. I found that there is indeed ion movement in the stem during protein translocation, thus suggesting that almost the entire voltage drop occurs across the Ф-clamp. Next, I studied the contribution of the six negatively charged residues in the channel lumen to the cation selectivity of the channel. By mutating each of these residues to serines, I observed that only a few of these anionic residues seem to have a significant impact on cation selectivity. Finally, I investigated the relationship between cation selectivity in the anthrax toxin channel and the kinetics of protein translocation. I concluded that macroscopic cation selectivity alone is not a reliable predictor of translocation rates; local changes in electrostatic forces must be considered as well.
Source: Dissertation Abstracts International, Volume: 77-03(E), Section: B.;Advisors: Alan Finkelstein.