Molecular mechanisms of anthrax toxin unfolding and translocation - PROJECT SUMMARY To function, a protein must be correctly localized in the cell, especially in ones that are internally compartmentalized by membrane bilayers. Proteinaceous, membrane-embedded transporters, called translocase channels, can traffic proteins across membranes by a process known as transmembrane protein translocation. Translocase channels also play key functional roles in microbial pathogenesis, because a host cell’s lipid bilayer membrane functions as a formidable, first line of defense, isolating the pathogen from its cytosol. The bacterium, Bacillus anthracis, for example, secretes a three-protein toxin, called anthrax toxin, which is composed of protective antigen (PA), lethal factor (LF), and edema factor (EF). PA assembles into a translocase channel, forming a narrow passageway across the host cell’s endosomal membrane bilayer. The channel, however, is so narrow that LF and EF traverse it as unfolded polypeptide chains potentially with some transient helical structure. Once inside the target cell’s cytosol, LF and EF refold and then catalyze reactions that disrupt the cell’s normal physiology. Studies of protein unfolding and transmembrane translocation probe exciting biophysical questions, which apply broadly to the studies of soluble molecular motors, which unfold, disassemble, and degrade proteins. How is a thermodynamically stable substrate protein unfolded in the cell? What structural features in the translocase channel determine the complex energy landscape that guides a chemically-complex, unfolded chain through the narrow confines of the channel? The biophysical chemistry of transmembrane protein translocation, nonetheless, has been challenging to characterize, and the three-dimensional structures of many translocase channels are unknown. Bacterial toxins, like anthrax toxin, are particularly well-suited for these studies, because they carry their own translocase-channel machinery, which is able to spontaneously insert into lipid bilayer membranes. We will couple the spectroscopic tools used to study how proteins fold and unfold with single-channel planar lipid bilayer electrophysiology. We are ultimately interested in how these systems function as proton-gradient driven ratchets, how the unfoldase active sites or polypeptide clamps stabilize unfolding intermediates, how these clamp sites gate and ungate. Our overall goal is to define the molecular mechanism of force transduction and ratchet-based unfolding and translocation. Relevance: Knowledge of protein translocation mechanisms are of practical importance not only to developing novel methods to neutralize the toxin but also to advancing technologies, which exploit toxins as delivery vehicles for heterologous antigens and cytotoxins into immune and cancer cells.
