Abstract
Botulinum neurotoxin (BoNT) has applications in cosmetic procedures and is used as a therapeutic, but perhaps it is the potential use as a biological weapon that captures the attention of most. BoNT can cause persistent paralysis at peripheral nerve terminals, by disrupting the release of neurotransmitters at the presynaptic motor nerve terminal. The metalloprotease domain targets the cytosolic SNARE substrates which are required for synaptic vesicle (SV) exocytosis. The targets of the metalloprotease are well characterised, however we still lack a detailed molecular picture of the translocation and trafficking mechanism of Botulinum neurotoxin type A (BoNT/A), in particular how it escapes synaptic vesicles. To understand this we have developed an in-vitro single-molecule assay of translocation. Using Droplet Interface Bilayers we observe single-molecule translocation events consistent with monomeric activity independent of channel formation.This thesis introduces Botulinum neurotoxin in Chapter 1 with a brief back-ground to what is currently known about its structure and function and what the accepted models of translocation are, it also contains a discussion on some com-mon models of membrane interfaces. The different constructs, experimental and analytical techniques used in this thesis are introduced in Chapter 2. Chapter 3 discusses our electrical and optical experiments at both bulk and single molecule concentrations, our findings in this chapter pointed to a differing mechanism than those previously reported, we then develop a two-part assay to detect translocation in Chapter 4 which is verified using other BoNT constructs. We discuss our Cryo-EM experiments of BoNT in SUVs in Chapter 5 before presenting our proposed model with concluding remarks in Chapter 6.
| Date of Award | 1 Jun 2024 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Mark Wallace (Supervisor) & Rivka Isaacson (Supervisor) |