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Title page for ETD etd-08252011-124331

Type of Document Dissertation
Author Mushrush, Darren J
URN etd-08252011-124331
Title Mechanistic details of the pH-dependent association of botulinum neurotoxin with membranes
Degree PhD
Department Biochemistry
Advisory Committee
Advisor Name Title
D. Borden Lacy Committee Chair
Charles R. Sanders Committee Member
Hassane S. Mchaourab Committee Member
Michael R. Waterman Committee Member
Richard N. Armstrong Committee Member
  • liposomes
  • EPR
  • fluorescence
  • bacterial toxin
  • biophysics
Date of Defense 2011-08-04
Availability unrestricted
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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