Single Reconstituted Neuronal SNARE Complexes Zipper in Three Distinct Stages

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Science  16 Aug 2012:
DOI: 10.1126/science.1224492

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SNARE proteins drive membrane fusion by assembling into a four-helix bundle in a zippering process. Here, we used optical tweezers to observe in a cell-free reconstitution experiment in real time a long-sought SNARE assembly intermediate in which only the membrane-distal N-terminal half of the bundle is assembled. Our findings support the zippering hypothesis, but suggest that zippering proceeds through three sequential binary switches, not continuously, in the N- and C-terminal halves of the bundle and the linker domain. The half-zippered intermediate was stabilized by externally applied force that mimicked the repulsion between apposed membranes being forced to fuse. This intermediate then rapidly and forcefully zippered, delivering free energy of 36 kBT to mediate fusion.

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