Quantitative characterization of neuronal trans-SNARE complexes using DNA origami

项目摘要/摘要 神经传递的一个关键步骤是突触囊泡(SV)膜与神经元的融合 质膜(PM)，将神经递质释放到突触间隙，在那里它们结合并激活POST 突触受体。一种名为SNARE的蛋白质复合体被认为发挥了核心作用，因为它的组装可以 产生足够的能量来驱动核聚变。目前描述圈套介导的融合的假设是 称为“SNARE Zippering”：SV上的v-SNARE蛋白与PM上的t-SNARE蛋白异源二聚体结合。 拉链状的方式，形成跨SNARE复合体(即v-和t-SNARE跨膜结构域 嵌入在单独的膜中)；释放的能量最终克服了 SV和PM并将这两种膜拉在一起，其中反式SNARE复合体转化为顺式SNARE 复合体(即v-和t-陷阱位于单一膜上)。目前所知的大部分关于 神经元陷阱的结构和动力学源于对顺式陷阱的分析，但真正的英雄是跨陷阱的 这为膜融合提供了推动力，但仍然难以捉摸。这里的一个主要技术挑战是 捕获在快速胞吐过程中形成的部分组装的反式SNARE复合体(&lt；1ms)。 在这个方案中，我们通过结合DNA的纳米级可编程性提供了一个解决方案 纳米技术和使用纳米盘(ND)限制熔融孔扩张的能力。一个V形DNA 折纸结构用于容纳两个结合部分；一个部分包括已被 在NDS中重组，而另一种则由NDS与同源T形圈套组成。我们的平台显著地 通过研究改进了以往揭示神经元跨SNARE组装真实信息的方法：(1) 全长SNARE蛋白，而不是截断或突变，因为拉链的中断是单独发生的 来自距离控制；(2)脂双层中的陷阱，代表它们的自然环境。 在特定目标1中，存在于双层中的一组部分组装的神经元反式SNARE复合体 它模仿突触融合机制中的渐进式四核。然后是各种梭状芽胞杆菌 神经毒素(CNTs)被添加到复杂集合中，与陷阱组装完备性的关系 并且可以系统地检测碳纳米管的蛋白降解活性。在特定目标2中，一种改进的V形折纸 作为一个力谱仪来研究神经元跨SNARE组装的能量图景 双层的背景。重要的是，我们将研究疾病相关的圈套突变对 跨复合体组装能量，这将有助于阐明它们对精神障碍的影响。 简而言之，我们努力建立一个新颖而强大的平台，重新审视这一中心但难以捉摸的机器之一 神经科学领域：神经元跨圈套复合体。关于广泛使用的碳纳米管的重要知识和 预计在这项研究中将获得与疾病相关的突变体，这可能使基础研究和 临床实践。这种基于DNA的技术也可以用于体外研究其他膜蛋白。

Project Summary/Abstract A key step in neurotransmission is the fusion of the synaptic vesicle (SV) membrane with neuronal plasma membrane (PM), to release neurotransmitters into the synaptic cleft where they bind and activate post synaptic receptors. A protein complex called SNARE is believed to play a central role since its assembly can generate enough energy to drive fusion. The current hypothesis that describes SNARE-mediated fusion is referred to as 'SNARE zippering': a v-SNARE protein on SV binds to a t-SNARE protein heterodimer on PM in a zipper-like fashion, forming a trans-SNARE complex (i.e. v- and t-SNARE transmembrane domains are embedded in separate membranes); the released energy eventually overcomes the repulsive forces between SV and PM and pulls the two membranes together, where trans-SNARE complexes transform into cis-SNARE complexes (i.e. v- and t-SNAREs locate on a single membrane). At present most of what is known concerning neuronal SNARE structure and dynamics stems from analysis of cis-SNARE, but the 'real hero' trans-SNARE that provides the driving force for membrane fusion remains elusive. A main technical challenge here is to capture partially assembled trans-SNARE complexes that form during the fast process of exocytosis (<1 ms). In this proposal, we offer a solution by combining the power of nanoscale programmability from DNA nanotechnology and the ability of restricting fusion pore expansion by using nanodisc (ND). A V-shaped DNA origami structure is used for hosting two binding moieties; one moiety comprises v-SNAREs that have been reconstituted in NDs, while the other comprises NDs with the cognate t-SNAREs. Our platform significantly improved previous methods in revealing true information of neuronal trans-SNARE assembly by studying: (1) full-length SNARE proteins rather than truncations or mutations, as the disruption of zippering solely arises from distance control; (2) SNAREs in lipid bilayers, which represent their native environment. In Specific Aim 1, a set of partially-assembled neuronal trans-SNARE complexes residing in bilayers are produced, which mimic the progressive quaternary core in synaptic fusion machinery. Then various clostridial neurotoxins (CNTs) are added into the complex set, and the relation between SNARE assembly completeness and CNTs' proteolytic activity could be systematically examined. In Specific Aim 2, a modified V-origami functions as a force spectrometer to investigate the energy landscape of neuronal trans-SNARE assembly in the context of bilayers. Importantly, we will examine the effect of disease-associated SNARE mutations on trans-complex assembly energy, which would help elucidate their impact on psychiatric disorders. In brief, we strive to build a novel and powerful platform to revisit one of the central yet elusive machinery in neuroscience: the neuronal trans-SNARE complex. Important knowledge concerning widely-used CNTs and disease-relevant mutants are expected to acquire in this study, potentially benefiting both basic research and clinical practices. Such DNA-based technology may also be used to study other membrane proteins in vitro.