Interdisciplinary Research Center HALOmem, Charles Tanford Protein Center, Institute for Biochemistry and Biotechnology, Martin Luther University Halle-Wittenberg, Kurt-Mothes-Str. 3a, 06120, Halle (Saale), Germany.
Institute of Chemistry and Biochemistry - Organic Chemistry, Freie Universität Berlin, Takustr. 3, 14195, Berlin, Germany.
J Am Soc Mass Spectrom. 2019 Jan;30(1):149-160. doi: 10.1007/s13361-018-2000-4. Epub 2018 Jun 12.
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.
突触融合蛋白 2 是神经元信号传递中的关键因子。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，并介导突触小泡与突触前膜的胞吐作用。虽然突触融合蛋白 2 是 SNARE 复合物中的四螺旋束的一部分，但在没有脂质或其他 SNARE 蛋白的情况下，它是天然无结构的。在先前的研究中，已经鉴定出部分折叠的片段，推测是 SNARE 复合物形成的中间体，以及突触融合蛋白 2 二聚体和寡聚体的形成。在这里，我们使用三种突触融合蛋白 2 变体——全长蛋白 Syb(1-116)、可溶性胞质变体 Syb(1-96)以及包含结构片段但缺少 SNARE 复合物形成触发位点的更短版本 Syb(49-96)——来研究在没有相互作用伙伴或整合到脂质体双层中的情况下寡聚化。将天然质谱与化学交联相结合，我们发现截短的变体显示出更高的寡聚化。我们从这两种技术中得到的发现非常吻合，并证实了溶液中存在寡聚物，而膜结合的突触融合蛋白 2 主要是单体。使用离子淌度质谱，我们可以进一步表明，Syb(49-96)寡聚物的较低电荷状态（可能代表溶液结构）遵循各向同性生长曲线，表明它们是固有无序的。从技术角度来看，我们表明天然离子淌度质谱与化学交联的结合非常适合分析蛋白质同型寡聚体。
