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    Mechanistic details of the pH-dependent association of botulinum neurotoxin with membranes

    Mushrush, Darren J
    : https://etd.library.vanderbilt.edu/etd-08252011-124331
    http://hdl.handle.net/1803/14006
    : 2011-09-06

    Abstract

    Botulinum neurotoxin (BoNT) belongs to a large class of toxic proteins that act by enzymatically modifying cytosolic substrates within eukaryotic cells. The process by which a catalytic moiety is transferred across a membrane to enter the cytosol is not understood for any such toxin. BoNT is known to form pH-dependent pores important for the translocation of the catalytic domain into the cytosol. As a first step toward understanding this process, we have investigated the mechanism by which the translocation domain of BoNT associates with a model liposome membrane. We report conditions that allow pH-dependent proteoliposome formation and identify a sequence at the translocation domain C-terminus that is protected from proteolytic degradation in the context of the proteoliposome. Fluorescence quenching experiments suggest that residues within this sequence move to a hydrophobic environment upon association with liposomes. Electron paramagnetic resonance analyses of spin labeled mutants reveal major conformational changes in a distinct region of the structure upon association and indicate the formation of an oligomeric membrane-associated intermediate. Together, these data support a model of how BoNT orients with membranes in response to low pH.
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