In the neuron, neurotransmitter release is mediated by SNARE (soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptor) proteins. SNARE-dependent synaptic vesicle membrane and plasma membrane fusion is a multiple-step event and a tightly regulated process. Vesicle-anchored (v-) SNARE from synaptic vesicles associates with target plasma membrane-anchored (t-) SNARE to form a trans-SNAREpin complex. When the triggering signal arrives, v-SNARE and t-SNARE mediate the membrane full fusion and extend on one side of the membrane, forming a cis-conformation. During the whole process, SNARE complex with the help of regulators overcomes the energy barriers to fuse two apposed membranes and ensures that fusion proceeds at the correct time and place.
Currently, there are some key questions that remain regarding SNARE-mediated exocytosis regulation. First, among the SNARE regulators, complexin is a small SNARE-binding protein that is thought to inhibit membrane fusion before Ca2+ triggering signal arrives. Although such an inhibitory role of complexin has been reported, its structural basis is very controversially discussed. Second, as the central machinery of neurotransmitters release, all three SNARE proteins are targets of different botulinum neurotoxins (BoNTs). Even though BoNT A and E cleave SNAP-25 at the C-terminus to inhibit SNARE-dependent membrane fusion, the detailed effects of BoNT A and E cleavage on SNARE complex folding pathway, conformation and function remain largely elusive. Third, the cis-SNARE complex contains 16 layers. BoNT E and A cleave SNAP-25 at residue 180 within layer \u27+2\u27 and residue 197 within layer \u27+7\u27, separately. The effect of SNAP-25 layers on SNARE complex formation has not been systematically studied. Also, another knowledge gap is why naturally selected BoNT E and A choose to cleave SNAP-25 at residue 180 and 197.
In this thesis, to solve the aforementioned questions, we primarily used single-molecule fluorescence resonance energy transfer (smFRET) to investigate the trans-SNAREpin and cis-SNARE complex formation and structure in the presence of SNARE regulators. Our results demonstrate that complexin splits the SNARE core in the C-terminal region to inhibit further SNARE zippering. We also conclude that the two membranes are necessary for the proper complexin function and are an integral part of the synaptic vesicle fusion regulatory machinery. SNAP-25E, the cleavage product by BoNT E, significantly decreases t- and v-SNARE pairing. The cleavage product by BoNT A SNAP-25A, however, does not affect the t- and v-SNARE pairing but mildly decreases SNARE zippering. In addition, our results unveil a delicate alpha-helix nucleation process at the SNAP-25 C-terminal motif (SC) downstream layers. The results also shed light on why BoNT E but not BoNT A can induce neuron degeneration
