Quantitative characterization of neuronal trans-SNARE complexes using DNA origami

项目概要/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），以释放神经递质进入突触间隙，在那里它们结合并激活后 突触受体一种名为SNARE的蛋白质复合物被认为起着核心作用，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SNARE介导融合的假说是 称为“SNARE拉链”：SV上的v-SNARE蛋白与PM上的t-SNARE蛋白异二聚体结合， 拉链样方式，形成反式SNARE复合物（即， 嵌入在单独的膜中）;释放的能量最终克服了 SV和PM并将两个膜拉在一起，在那里反式SNARE复合物转化为顺式SNARE 复合物（即v-和t-SNARE位于单个膜上）。目前，大多数人都知道， 神经元SNARE的结构和动力学源于顺式SNARE的分析，但“真实的英雄”反式SNARE 为膜融合提供驱动力的机制仍然是难以捉摸的。这里的主要技术挑战是 捕获在胞吐作用的快速过程（<1 ms）期间形成的部分组装的反式SNARE复合物。 在这个建议中，我们提供了一个解决方案，结合DNA的纳米级可编程能力， 纳米技术和通过使用纳米盘（ND）限制熔融孔膨胀的能力。V形DNA 折纸结构用于容纳两个结合部分;一个部分包含已被 在ND中重构，而另一种包含具有同源t-SNARE的ND。我们的平台显著 改进了以往揭示神经元trans-SNARE组装真实信息的方法，通过研究：（1） 全长SNARE蛋白，而不是截短或突变，因为拉链的破坏只会产生 （2）脂质双层中的SNARE，这代表了它们的天然环境。 在具体目标1中，一组位于双层中的部分组装的神经元反式SNARE复合物被 产生，其模仿突触融合机制中的渐进式四元核心。然后各种梭菌 神经毒素（CNT）被添加到复杂的集合，和SNARE组装完整性之间的关系 并可系统地检测碳纳米管的蛋白水解活性。在《特定目标2》中，一种改良的V形折纸 作为一个力谱仪的功能，以调查神经元trans-SNARE组装的能量景观， 双层的背景。重要的是，我们将研究疾病相关SNARE突变对 这将有助于阐明它们对精神疾病的影响。 简而言之，我们努力建立一个新颖而强大的平台，重新审视一个核心但难以捉摸的机制， 神经元反式陷阱复合物。关于广泛使用的碳纳米管的重要知识， 疾病相关的突变体预计将在这项研究中获得，可能有利于基础研究和 临床实践。这种基于DNA的技术也可用于在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.