The two enzymatic components of anthrax toxin, lethal factor (LF) and edema factor (EF), are transported to the cytosol of mammalian cells by the third component, protective antigen (PA). A heptameric form of PA binds LF and/or EF and, under the acidic conditions encountered in endosomes, generates a membrane-spanning pore that is thought to serve as a passageway for these enzymes to enter the cytosol. The pore contains a 14-stranded transmembrane b-barrel that is too narrow to accommodate a fully folded protein, necessitating that LF and EF unfold, at least partly, in order to pass. Here, we describe the pH-dependence of the unfolding of LF N and EF N , the 30 kDa N-terminal PA-binding domains, and minimal translocatable units, of LF and EF. Equilibrium chemical denaturation studies using fluorescence and circular dichroism spectroscopy show that each protein unfolds via a fourstate mechanism: N4I4J4U. The acid-induced N/I transition occurs within the pH range of the endosome (pH 5-6). The I state predominates at lower pH values, and the J and U states are populated significantly only in the presence of denaturant. The I state is compact and has characteristics of a molten globule, as shown by its retention of significant secondary structure and its ability to bind an apolar fluorophore. The N/I transition leads to an overall 60% increase in buried surface area exposure. The J state is expanded significantly and has diminished secondary structure content. We analyze the different protonation states of LF N and EF N in terms of a linked equilibrium proton binding model and discuss the implications of our findings for the mechanism of acidic pH-induced translocation of anthrax toxin. Finally, analysis of the structure of the transmembrane b-barrel of PA shows that it can accommodate a-helix, and we suggest that the steric constraints and composition of the lumen may promote a-helix formation
