Interactions of quinolinols, peptides and SNAP-25 with the protease domain of botulinum neurotoxin A
Thesis (Ph.D.)--Georgetown University, 2010.; Includes bibliographical references.; Text (Electronic thesis) in PDF format. SNAP-25, an intrinsically disordered protein, assembles with syntaxin and synaptobrevin to form a SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex, a four-helix bundle that plays a key role in the exocytosis of synaptic vesicles. Botulinum neurotoxins specifically cleave one of the SNARE proteins, resulting in the inhibition of membrane fusion. Small molecule inhibitors are being evaluated as potential therapeutic counter-measurement to the toxin's deleterious action on the neuromuscular junctions.; In order to understand the conformational changes exhibited by SNAP-25 prior to and during SNARE complex formation, and its biological function, we developed a method of investigating the conformational changes of SNAP-25 in the absence and presence of Zn2+-depleted BoNT/A LC (botulinum neurotoxin A light chain), using FRET (fluorescence resonance energy transfer) between EGFP (enhanced green fluorescent protein) and CCPGCC-ReAsH fused to SNAP-25 at specific locations. The apparent distances between the C-terminus and the N-terminus, the C-terminus and the D140, and the N-terminus and the D140 of the SNAP-25 protein were determined based on the FRET efficiency in the absence and presence of Zn2+-depleted BoNT/A LC. Various mutants of SNAP-25(A195S) were genetically engineered and used to measure the tertiary structures of SNAP-25(A195S) in the absence and presence of active BoNT/A LC. Shorter apparent distances in the presence of BoNT/A LC suggest that the SNAP-25 folds around BoNT/A LC in the binary complex. FRET is a useful tool to investigate the conformational changes of other intrinsically disordered proteins.
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Sultani, Mashal (Georgetown University, 2012)Botulism neurotoxins (BoNTs), a family of neurotoxins produced by the anaerobic bacterium Clostridium botulinum, are the most poisonous biological toxins known to man and are listed as category A bioterrorism agents by the ...