anthrax toxin protective antigen inserts into and delivers one of two catalytic factors across a membrane bilayer.
The specific aims are to first elucidate the three-dimensional structure of the anthrax protective antigen in its membrane-spanning state using x- ray crystallography. This structure along with mutagenesis data will identify the molecular basis for acid-triggered pore formation. Extending the study to translocation involves first solving the structure of edema factor, alone or in complex with protective antigen. This structure will serve as the template for a rational incorporation of cross-links to study the requirement of unfolding in translocation. These experiments should allow for the identification of the minimal structural requirements for pore formation and the interactions required for both binding of the catalytic factor and its translocation. In addition to the health applications associated with understanding the role of the toxins in the anthrax disease symptoms, a molecular understanding of this process of pore formation and passenger protein translocation across membrane bilayers can be applied to other toxin systems, secretion systems, and mitochondrial import/export processes. Further, anthrax has recently been shown to have application in the delivery of heterologous proteins for vaccine development. The methods employed in this study will include x-ray crystallography, site directed mutagenesis, and both in vitro and in vivo assays for pore formation and translocation.
