The SNARE hypothesis states that the folding and assembly of four-helix SNARE complex bundles drives intracellular membrane fusion, including fusion in exocytosis of synaptic vesicles at the plasma membranes of synaptic terminals in neurons. The neuronal SNAREs are syntaxin 1 a and SNAP-25 on the plasma membrane and synaptobrevin 2 on the vesicle membrane. Syntaxin contributes one and SNAP-25 contributes two helices to the active acceptor SNARE complex on the target membrane. Synaptic vesicles dock to the presynaptic plasma membrane by contributing synaptobrevin to the nascent SNARE complex. How far this trans-SNARE complex that bridges the two membranes is folded is presently not known. The trans-SNARE complex is then thought to zipper-fold from the N- towards the C-terminus of the parallel fourhelix bundle. The bundle, thereby pulling the two membranes into close proximity. In a final step, SNARE complex folding proceeds into the transmembrane domains of syntaxin and synaptobrevin to form a cis- SNARE complex, i.e. a step which must be coupled with merging the lipid bilayers of the two membranes. The research proposed in this project will dissect the described folding steps and correlate them with different stages of vesicle docking and membrane fusion. Magnetic resonance approaches will be taken to determine (dynamic) structures of SNARE folding intermediates and micro-fluorescence approaches will be taken to determine the kinetics and higher architecture of the evolving fusion pore in artificial and cellular membrane preparations.

Public Health Relevance

Fusion of synaptic vesicles with the presynaptic membrane of neurons is a key element of neurotransmitter release in synaptic transmission. Defects in synaptic transmission lead to epilepsy, depression, and other neurological disorders. The research of this grant will elucidate the basic mechanism that leads to presynaptic membrane fusion and thus help define disorders in which this mechanism is disturbed.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Program Projects (P01)
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Special Emphasis Panel (ZRG1-BCMB-S (40))
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University of Virginia
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Witkowska, Agata; Jahn, Reinhard (2017) Rapid SNARE-Mediated Fusion of Liposomes and Chromaffin Granules with Giant Unilamellar Vesicles. Biophys J 113:1251-1259
Kreutzberger, Alex J B; Kiessling, Volker; Liang, Binyong et al. (2017) Asymmetric Phosphatidylethanolamine Distribution Controls Fusion Pore Lifetime and Probability. Biophys J 113:1912-1915
Katti, Sachin; Nyenhuis, Sarah B; Her, Bin et al. (2017) Non-Native Metal Ion Reveals the Role of Electrostatics in Synaptotagmin 1-Membrane Interactions. Biochemistry 56:3283-3295
Kreutzberger, Alex J B; Kiessling, Volker; Liang, Binyong et al. (2017) Reconstitution of calcium-mediated exocytosis of dense-core vesicles. Sci Adv 3:e1603208
Zdanowicz, Rafal; Kreutzberger, Alex; Liang, Binyong et al. (2017) Complexin Binding to Membranes and Acceptor t-SNAREs Explains Its Clamping Effect on Fusion. Biophys J 113:1235-1250
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Liang, Binyong; Tamm, Lukas K (2016) NMR as a tool to investigate the structure, dynamics and function of membrane proteins. Nat Struct Mol Biol 23:468-74
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Milovanovic, Dragomir; Platen, Mitja; Junius, Meike et al. (2016) Calcium Promotes the Formation of Syntaxin 1 Mesoscale Domains through Phosphatidylinositol 4,5-Bisphosphate. J Biol Chem 291:7868-76

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