Fasshauer D, Otto H, Eliason W K, Jahn R, Brünger A T
Howard Hughes Medical Institute, New Haven, Connecticut 06510, USA.
J Biol Chem. 1997 Oct 31;272(44):28036-41. doi: 10.1074/jbc.272.44.28036.
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 ternary complex that can be dissociated by the ATPase N-ethylmaleimide-sensitive 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 studies aimed at elucidating such changes in vitro using recombinant proteins. All proteins were purified in a monomeric form. Syntaxin showed significant alpha-helicity, whereas SNAP-25 and synaptobrevin exhibited characteristics of largely unstructured proteins. Formation of the ternary complex induced dramatic increases in alpha-helicity and in thermal 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 ternary complex. We propose that the transition from largely unstructured monomers to a tightly packed, energetically favored ternary complex connecting two membranes is a key step in overcoming energy barriers for membrane fusion.
突触小体相关蛋白25（SNAP - 25）、 syntaxin和突触囊泡蛋白在突触小泡的调节性胞吐作用中起关键作用，但其作用机制尚不清楚。在体外，这些蛋白质能自发组装成三元复合物，该复合物可被ATP酶N - 乙基马来酰亚胺敏感融合蛋白以及辅因子α - 、β - 和γ - SNAP解离。由于与这些反应相关的结构变化可能构成膜融合的基础，我们已着手进行生物物理研究，旨在利用重组蛋白在体外阐明此类变化。所有蛋白质均以单体形式纯化。Syntaxin表现出显著的α螺旋性，而SNAP - 25和突触囊泡蛋白则表现出大部分为无结构蛋白的特征。三元复合物的形成导致α螺旋性和热稳定性显著增加。这表明在复合物形成时，SNAP - 25和突触囊泡蛋白会诱导形成结构。此外，化学计量从Syntaxin - SNAP - 25复合物中的2:1变为三元复合物中的1:1:1。我们提出，从大部分无结构的单体转变为紧密堆积、能量有利的连接两个膜的三元复合物是克服膜融合能量障碍的关键步骤。
