The long term objectives of this project are two-fold: (1) to develop detailed molecular models which fully account for the formation and voltage dependence of channels formed in membranes by several bacterial proteins; (2) to determine the mechanism by which the channel-forming regions of these proteins translocate other regions of themselves, or other protein, across the membrane. The methodology for achieving these goals is the study in planar phospholipid bilayer membranes of voltage gating of these channels and the protein translocation associated with that gating. The channels focused on are those formed by colicin la, the T-domain of diphtheria toxin (DT) and the PA63 -component of anthrax toxin. With respect to the first objective: mutations will be made of charged residues in the channel- forming regions of colicin la and DT in order to identify their voltage-sensing elements. Chimeras of these two proteins will also be constructed in the hope of assigning specific channel properties (e.g., conductance, pH sensitivity, and voltage gating) to specific protein segments. With respect to the second objective: massive translocation of protein occurs across planar lipid bilayers in association with the colicin la and DT channels; whether these channels are conduits for protein translocation or if instead protein translocation is coupled to the channel formation process itself will be determined. The channel formed by the PA63-component of anthrax toxin is the conduit for protein translocation, and the parameters controling translocation will be investigated. The elucidation of how the channel-forming domains of the toxins under study are involved in the translocation of the enzymatic (killing) domains to the cytosol should be of great help in the effective design of chimeric proteins that can function as """"""""magic bullets"""""""" in the killing of cancer cells. In addition, the development of agents that can block the PA63 channel of anthrax toxin could prove useful as a defense against this potential terrorist weapon.
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