Molecular mechanisms of protein transport through the anthrax toxin channel
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The toxin produced by Bacillus anthracis, the causative agent of anthrax, is a model system to study the mechanisms of protein translocation across biological membranes. The toxin is composed of a translocase channel formed from protective antigen (PA), which allows its two substrate proteins, lethal and edema factors (LF and EF), to translocate to the host cell cytosol causing cell damage. The PA incorporated into planar lipid bilayers forms a cation-selective channel capable of transporting EF, LF, and LFN (the N-terminal 263 residues of LF, on which our experiment were done). Protein translocation through the channel is driven by a proton electrochemical potential gradient, on a time scale of seconds, and does not require any additional cellular proteins. In this thesis work, we showed: (i) that the intrinsic kinetics of LFN translocation are S-shaped; that is, they are not solely due to the fact multiple LFNs are translocated in tandem. (ii) that if only one non-titratable SO3 - group is introduced at most position in LFN, there is a dramatic reduction in the LFN translocation rate. This result is consistent, with the PA channel strongly disfavoring the entry of negatively charged residues on proteins to be translocated. We also found that a major barrier to anion entry to the channel occurs at the phi-clamp, a ring of seven phenylalanines near the channel entrance. We proposed a proton-protein symporter mechanism; that is, the acidic groups on LFN enter protonated (i.e. neutralized), and upon exiting the channel, the protons that were picked up from the cis solution are released into the trans solution. (iii) Given that the channel stem is 100 A-long, 15 A-wide, proteins must unfold to pass through, but it is not clear if the secondary structure is preserved. To address this question, we developed a technique to trap a translocating polypeptide chain of a protein within the PA channel. We determined the minimum number of LFN residues required to span the length of the channel. We concluded that LFN traverses the channel as an extended chain, and that the channel unfolds the alpha-helices of LFN as it is being translocated.