Single-Molecule Optical Tweezers Study of Regulated SNARE Assembly
Intracellular membrane fusion mediates material and information exchange among different cells or cellular compartments with high accuracy and spatiotemporal resolution. Fusion is driven by ordered folding and assembly of soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP) receptors (SNAREs) and regulated by many other proteins. Understanding regulated SNARE assembly is key to dissecting mechanisms and physiologies of various fusion processes and their associated diseases. Yet, it remains challenging to study regulated SNARE assembly using traditional ensemble-based experimental approaches. Here, we describe our new method to measure the energy and kinetics of neuronal SNARE assembly in the presence of α-SNAP, using a single-molecule manipulation approach based on high-resolution optical tweezers. Detailed experimental protocols and methods of data analysis are shown. This approach can be widely applied to elucidate the effects of regulatory proteins on SNARE assembly and membrane fusion.
Key wordsOptical tweezers Single-molecule manipulation SNARE assembly and disassembly SNAP NSF Hidden Markov modeling Energy landscape
This work was supported by NIH grants RO1GM093341 and RO1GM120193 to Y.Z. We also thank the support from the National Natural Science Foundation of China (31770812 to M.L.) and the Chinese Academy of Science Chengkun Wang talent program and Jiaxi Lu international team.
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