STRUCTURAL-CHANGES ARE ASSOCIATED WITH SOLUBLE N-ETHYLMALEIMIDE-SENSITIVE FUSION PROTEIN ATTACHMENT PROTEIN-RECEPTOR COMPLEX-FORMATION

SNAP-25, syntaxin, and synaptobrevin play a key role in the regulated exocytosis of synaptic vesicles, but their mechanism of action is not understood. In vitro, the proteins spontaneously assemble into a terna ry complex that can be dissociated by the ATPase N-ethylmaleimide-sens itive fusion protein and the cofactors alpha-, beta-, and gamma-SNAP. Since the structural changes associated with these reactions probably form the basis of membrane fusion, we have embarked on biophysical stu dies aimed at elucidating such changes in vitro using recombinant prot eins. All proteins were purified in a monomeric form. Syntaxin showed significant alpha-helicity, whereas SNAP-25 and synaptobrevin exhibite d characteristics of largely unstructured proteins. Formation of the t ernary complex induced dramatic increases in alpha-helicity and in the rmal stability. This suggests that structure is induced in SNAP-25 and synaptobrevin upon complex formation. In addition, the stoichiometry changed from 2:1 in the syntaxin-SNAP-25 complex to 1:1:1 in the terna ry complex. We propose that the transition from largely unstructured m onomers to a tightly packed, energetically favored ternary complex con necting two membranes is a key step in overcoming energy barriers for membrane fusion.