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Science 337 (6100): 1340-1343

Copyright © 2012 by the American Association for the Advancement of Science

Single Reconstituted Neuronal SNARE Complexes Zipper in Three Distinct Stages

Ying Gao, Sylvain Zorman, Gregory Gundersen, Zhiqun Xi, Lu Ma, George Sirinakis, James E. Rothman,* Yongli Zhang*

Abstract: Soluble N-ethylmaleimide–sensitive factor attachment protein receptor (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 amino-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 amino- and carboxyl-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 (where kB is Boltzmann’s constant and T is temperature) to mediate fusion.

Department of Cell Biology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520, USA.

* To whom correspondence should be addressed. E-mail: yongli.zhang{at}yale.edu (Y.Z.); james.rothman{at}yale.edu (J.E.R.)

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