Single reconstituted neuronal SNARE complexes zipper in three distinct stages

Science. 2012 Sep 14;337(6100):1340-3. doi: 10.1126/science.1224492. Epub 2012 Aug 16.

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 k(B)T (where k(B) is Boltzmann's constant and T is temperature) to mediate fusion.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Cell-Free System
  • DNA / chemistry
  • DNA / metabolism
  • Entropy
  • Neurons / metabolism
  • Optical Tweezers*
  • Qa-SNARE Proteins / chemistry
  • SNARE Proteins / chemistry*
  • Vesicle-Associated Membrane Protein 2 / chemistry

Substances

  • Qa-SNARE Proteins
  • SNARE Proteins
  • Vesicle-Associated Membrane Protein 2
  • DNA