| Type of Document |
Dissertation |
| Author |
Mushrush, Darren
|
| 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 |
|
| Keywords |
- fluorescence
- EPR
- liposomes
- bacterial toxin
- biophysics
|
| Date of Defense |
2011-08-04 |
| Availability |
unrestricted |
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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| Files |
| Filename |
Size |
Approximate Download Time
(Hours:Minutes:Seconds)
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56K Modem |
ISDN (64 Kb) |
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Mushrush.pdf |
3.60 Mb |
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