Soluble N-ethylmaleimide-sensitive attachment receptor (SNARE) proteins are crucial for membrane fusion, assembling into a stable four-helix bundle that brings membranes close together. This process is driven by the energy released during complex formation. The assembly of SNARE complexes is regulated by several proteins, including Complexin, which partially associates with the core complex, potentially stabilizing intermediates and dissociating upon calcium trigger. However, Complexin's precise role remains debated. After fusion, the recycling of SNARE proteins is facilitated by the AAA+ protein NSF and its cofactor a-SNAP, making SNAREs available for subsequent fusion events. Currently, effective models to inhibit or slow down the disassembly mechanism are lacking. Research aimed to develop a potent inhibitor of the a-SNAP/NSF-mediated disassembly, utilizing the SNARE motif of Synaptobrevin as a model to explore SNARE/SNAP complex recognition sites. Various modifications were introduced to identify key interactions that could prevent a-SNAP recognition. Additionally, a ß-peptide analog of Complexin was synthesized to inhibit disassembly by enhancing interactions with Synaptobrevin and Syntaxin helices. The study also involved synthesizing small independently folding protein domains, labeled for single molecule fluorescence spectroscopy (smFRET), to examine conformational dynamics and interactions of proteins, providing insig
