PROJECT SUMMARY/ABSTRACT Complex behaviors of the brain, such as cognition, perception, motivation, and mental illness, still remain difficult to explain. To truly understand these processes, it is necessary to understand the basic mechanisms that underly them. Synaptic transmission, the release of neurotransmitters from the presynaptic neuron upon membrane fusion, relies on SNAREs (soluble N-ethylmaleimide sensitive factor attachment protein receptors). SNAREs on the neurotransmitter containing vesicles form a stable, trans SNARE complex with SNAREs on the presynaptic membrane. Once signaled, these SNAREs twist together to provide the energy necessary for membrane fusion. This cis SNARE complex, now a highly stable four helix bundle on one membrane, must be disassembled and recycled to allow further rounds of fusion. Without a pool of fusogenic SNAREs, synaptic transmission would cease. cis SNARE disassembly is accomplished by NSF (N- ethylmaleimide sensitive factor) and adaptor proteins called SNAPs (soluble NSF attachment proteins). Together, the three components form a 20S complex, in which NSF, upon ATP hydrolysis, disassembles SNARE complex and maintains a pool of fusogenic SNAREs. Yet the key dynamical processes and principles of this explosive disassembly step remain unknown. The overall goal of this project is to elucidate the fundamental mechanisms of synaptic transmission by understanding SNARE disassembly. To uncover the principles of SNARE disassembly, both NSF and its yeast ortholog Sec18 will be examined. Studying the dynamics of NSF in its neuronal context has proven difficult due to the complexity of the presynaptic system and the inability to investigate more than a handful of mutants at a time. Studying Sec18 and the yeast 20S (Y20S), in coordination with the neuronal 20S, will enable the use of a wide variety of molecular and biochemical tools that will allow for the dissection of NSF/Sec18 action. The high degree of orthology between the Y20S and 20S also means that observations and principles gained by studying the Y20S will directly transferrable to the neuronal 20S. The hypothesis is that disassembly of SNAREs by the Y20S is mediated by a conserved allosteric network that spans multiple promoters within the Y20S complex (and therefore the 20S complex as well), which play a key role in the modulation of neurotransmission. To test this hypothesis, CryoEM studies of Sec18 and the Y20S have already been completed. This has allowed for the determination of residues that correlate to differences in conformation, assisted by unsupervised machine learning methods. I propose saturation mutagenesis of every single residue in Sec18 in an in vivo assay tying Sec18 activity to survival that will reveal the fitness of each residue in its ability to mediate SNARE disassembly. Second, electrophysiology experiments on mutant NSF in key residues in this allosteric network will directly tie these biophysical mechanisms directly to synaptic...