Mechanistic Investigation of Membrane Fusion through a Model Caged SNARE Protein

Intracellular membrane fusion is directed by the formation of a specific complex of proteins such as SNARE [Soluble NSF (N-ethylmaleimide-sensitive factor) Attachment Protein Receptor]. There are several mechanisms of SNARE mediated membrane fusion, but the exact nature of these processes remains debated. In particular, little is understood about the molecular mechanisms governing trans-SNARE complex nucleation and zippering in driving fusion. Assembly strength and fusion kinetics in these systems are highly complex, and the downstream events of membrane contact (docking), stalk formation, hemifusion, and fusion pore opening along the fusion pathway is still unclear. Moreover, the exact role of the transmembrane domain (TMD) of synaptobrevin and syntaxin-1A is unknown. In this presentation, I will demonstrate light triggered mechanistic investigation of membrane fusion using artificial caged SNAREs. Caging of a biologically active molecule with a photolabile protecting group at a key functional position can temporarily mask the functionality and inactivate the biomolecule. The activity of the molecule can be restored by uncaging, externally triggered by light of appropriate wavelength. However, caging group strategy has not been applied yet to temporarily block the membrane fusion activity of SNARE protein to get deeper structural insights into the SNARE zippering and assembly pathway. I will illustrate the design of artificial caged SNAREs with photolabile protecting groups to dissect the mechanism of SNAREs in membrane docking, hemifusion, and fusion. I will also provide a method to arrest and study the intermediates such as partially zipped trans-SNARE complexes which is used for light triggered stepwise recognition/two-stage zippering of membrane fusion. Our recent findings of light triggered membrane fusion using artificial caged SNAREs can be a significant starting point to address many compelling questions surrounding the topic of SNARE-induced membrane fusion.

At the end, I will present my future research proposal: 1) Synthetic Transmembrane Peptide-Based Ion Channel and 2) Synthetic Molecular/Supramolecular Machines.