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, but the channel is so narrow that LF and EF traverse it as unfolded polypeptide chains. 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 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, however, has been challenging to characterize, and the three-dimensional structures of many translo- case 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 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.

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The scope of this application covers a structure/function study of the problem of cellular protein unfolding and transport. We will focus on anthrax toxin, a three- protein, bacterial toxin secreted by Bacillus anthracis. We are seeking to obtain a biophysical understanding of the toxin's transmembrane translocation mechanism, which allows its cytotoxic cargo to enter into mammalian host cells.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
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Biochemistry and Biophysics of Membranes Study Section (BBM)
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Breen, Joseph J
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University of California Berkeley
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Abrami, Laurence; Brandi, Lucia; Moayeri, Mahtab et al. (2013) Hijacking multivesicular bodies enables long-term and exosome-mediated long-distance action of anthrax toxin. Cell Rep 5:986-96
Feld, Geoffrey K; Brown, Michael J; Krantz, Bryan A (2012) Ratcheting up protein translocation with anthrax toxin. Protein Sci 21:606-24
Feld, Geoffrey K; Kintzer, Alexander F; Tang, Iok I et al. (2012) Domain flexibility modulates the heterogeneous assembly mechanism of anthrax toxin protective antigen. J Mol Biol 415:159-74
Sterling, Harry J; Kintzer, Alexander F; Feld, Geoffrey K et al. (2012) Supercharging protein complexes from aqueous solution disrupts their native conformations. J Am Soc Mass Spectrom 23:191-200
Brown, Michael J; Thoren, Katie L; Krantz, Bryan A (2011) Charge requirements for proton gradient-driven translocation of anthrax toxin. J Biol Chem 286:23189-99
Thoren, Katie L; Krantz, Bryan A (2011) The unfolding story of anthrax toxin translocation. Mol Microbiol 80:588-95
Sterling, Harry J; Cassou, Catherine A; Trnka, Michael J et al. (2011) The role of conformational flexibility on protein supercharging in native electrospray ionization. Phys Chem Chem Phys 13:18288-96
Kintzer, Alexander F; Sterling, Harry J; Tang, Iok I et al. (2010) Anthrax toxin receptor drives protective antigen oligomerization and stabilizes the heptameric and octameric oligomer by a similar mechanism. PLoS One 5:e13888
Kintzer, Alexander F; Sterling, Harry J; Tang, Iok I et al. (2010) Role of the protective antigen octamer in the molecular mechanism of anthrax lethal toxin stabilization in plasma. J Mol Biol 399:741-58
Feld, Geoffrey K; Thoren, Katie L; Kintzer, Alexander F et al. (2010) Structural basis for the unfolding of anthrax lethal factor by protective antigen oligomers. Nat Struct Mol Biol 17:1383-90

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