Single-molecule studies of SNARE complex assembly reveal parallel and antiparallel configurations
Single-molecule studies of SNARE complex assembly reveal parallel and antiparallel configurations
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DOI：
10.1073/pnas.2036428100
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发表时间：
2003-12-09
影响因子：
11.1
通讯作者：
Brunger, AT
中科院分区：
文献类型：
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作者：
Weninger, K;Bowen, ME;Brunger, AT
真核生物中的囊泡融合被认为涉及被称为可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体（SNARE）的高度保守的蛋白质家族组装成高度稳定的平行四螺旋束。我们已经使用了分子间单分子荧光共振能量转移表征预组装的神经元陷阱复合物组成的syntaxin，synaptobrevin，和突触体相关蛋白的25 kDa的沉积脂质双层。令人惊讶的是，我们发现了一个混合物的平行以及反平行的配置涉及陷阱图案的syntaxin和synaptobrevin以及那些syntaxin和突触相关蛋白的25 kDa。具有平行四螺旋束构型的亚群可以通过在变性剂存在下的额外纯化步骤而大大富集，表明平行构型是能量上最有利的状态。未观察到构型之间的相互转化。根据这一观察，我们推断转化率为
Vesicle fusion in eukaryotes is thought to involve the assembly of a highly conserved family of proteins termed soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) into a highly stable parallel four-helix bundle. We have used intermolecular single-molecule fluorescence resonance energy transfer to characterize preassembled neuronal SNARE complexes consisting of syntaxin, synaptobrevin, and synaptosome-associated protein of 25 kDa on deposited lipid bilayers. Surprisingly, we found a mixture of parallel as well as antiparallel configurations involving the SNARE motifs of syntaxin and synaptobrevin as well as those of syntaxin and synaptosome-associated protein of 25 kDa. The subpopulation with the parallel four-helix bundle configuration could be greatly enriched by an additional purification step in the presence of denaturant, indicating that the parallel configuration is the energetically most favorable state. Interconversion between the configurations was not observed. From this observation, we infer the conversion rate to be