Synaptobrevin-2 is a key player in signal transmission in neurons. It forms, together with SNAP25 and Syntaxin-1A, the neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex and mediates exocytosis of synaptic vesicles with the pre-synaptic membrane. While Synaptobrevin-2 is part of a four-helix bundle in this SNARE complex, it is natively unstructured in the absence of lipids or other SNARE proteins. Partially folded segments, presumably SNARE complex formation intermediates, as well as formation of Synaptobrevin-2 dimers and oligomers, were identified in previous studies. Here, we employ three Synaptobrevin-2 variants-the full-length protein Syb(1-116), the soluble, cytosolic variant Syb(1-96) as well as a shorter version Syb(49-96) containing structured segments but omitting a trigger site for SNARE complex formation-to study oligomerisation in the absence of interaction partners or when incorporated into the lipid bilayer of liposomes. Combining native mass spectrometry with chemical cross-linking, we find that the truncated versions show increased oligomerisation. Our findings from both techniques agree well and confirm the presence of oligomers in solution while membrane-bound Synaptobrevin-2 is mostly monomeric. Using ion mobility mass spectrometry, we could further show that lower charge states of Syb(49-96) oligomers, which most likely represent solution structures, follow an isotropic growth curve suggesting that they are intrinsically disordered. From a technical point of view, we show that the combination of native ion mobility mass spectrometry with chemical cross-linking is well-suited for the analysis of protein homo-oligomers. Graphical Abstract ᅟ.

中文翻译：

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通过天然质谱和化学交联研究Synaptobrevin-2的低聚。

Synaptobrevin-2是神经元信号传递的关键因素。它与SNAP25和Syntaxin-1A一起形成神经元可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体（SNARE）复合物，并介导突触前囊泡与突触前膜的胞吐作用。虽然Synaptobrevin-2是此SNARE复杂分子中四螺旋束的一部分，但在没有脂质或其他SNARE蛋白的情况下，它天然是非结构化的。在先前的研究中鉴定出部分折叠的片段，大概是SNARE复合物形成中间体，以及Synaptobrevin-2二聚体和寡聚体的形成。在这里，我们采用了三种Synaptobrevin-2变体-全长蛋白Syb（1-116），胞质变体Syb（1-96）以及包含结构化片段但却省略了SNARE复合物形成触发位点的较短版本Syb（49-96）-用于研究在缺乏相互作用伴侣或掺入脂质双分子层的情况下的寡聚脂质体。结合自然质谱与化学交联，我们发现截短的版本显示增加的低聚。我们从这两种技术中得出的结果都非常吻合，并证实了溶液中存在寡聚体，而膜结合的Synaptobrevin-2主要是单体。使用离子迁移质谱，我们可以进一步显示Syb（49-96）低聚物的较低电荷态（最有可能代表溶液结构）遵循各向同性生长曲线，表明它们本质上是无序的。从技术角度来看，我们表明，天然离子迁移质谱与化学交联相结合非常适合蛋白质均聚物的分析。图形摘要ᅟ。