Project Summary/Abstract A key step in neurotransmission is the fusion of the synaptic vesicle (SV) membrane with neuronal plasma membrane (PM), to release neurotransmitters into the synaptic cleft where they bind and activate post synaptic receptors. A protein complex called SNARE is believed to play a central role since its assembly can generate enough energy to drive fusion. The current hypothesis that describes SNARE-mediated fusion is referred to as 'SNARE zippering': a v-SNARE protein on SV binds to a t-SNARE protein heterodimer on PM in a zipper-like fashion, forming a trans-SNARE complex (i.e. v- and t-SNARE transmembrane domains are embedded in separate membranes); the released energy eventually overcomes the repulsive forces between SV and PM and pulls the two membranes together, where trans-SNARE complexes transform into cis-SNARE complexes (i.e. v- and t-SNAREs locate on a single membrane). At present most of what is known concerning neuronal SNARE structure and dynamics stems from analysis of cis-SNARE, but the 'real hero' trans-SNARE that provides the driving force for membrane fusion remains elusive. A main technical challenge here is to capture partially assembled trans-SNARE complexes that form during the fast process of exocytosis (<1 ms). In this proposal, we offer a solution by combining the power of nanoscale programmability from DNA nanotechnology and the ability of restricting fusion pore expansion by using nanodisc (ND). A V-shaped DNA origami structure is used for hosting two binding moieties; one moiety comprises v-SNAREs that have been reconstituted in NDs, while the other comprises NDs with the cognate t-SNAREs. Our platform significantly improved previous methods in revealing true information of neuronal trans-SNARE assembly by studying: (1) full-length SNARE proteins rather than truncations or mutations, as the disruption of zippering solely arises from distance control; (2) SNAREs in lipid bilayers, which represent their native environment. In Specific Aim 1, a set of partially-assembled neuronal trans-SNARE complexes residing in bilayers are produced, which mimic the progressive quaternary core in synaptic fusion machinery. Then various clostridial neurotoxins (CNTs) are added into the complex set, and the relation between SNARE assembly completeness and CNTs' proteolytic activity could be systematically examined. In Specific Aim 2, a modified V-origami functions as a force spectrometer to investigate the energy landscape of neuronal trans-SNARE assembly in the context of bilayers. Importantly, we will examine the effect of disease-associated SNARE mutations on trans-complex assembly energy, which would help elucidate their impact on psychiatric disorders. In brief, we strive to build a novel and powerful platform to revisit one of the central yet elusive machinery in neuroscience: the neuronal trans-SNARE complex. Important knowledge concerning widely-used CNTs and disease-relevant mutants are expected to acqu...