Quantitative characterization of neuronal trans-SNARE complexes using DNA origami

使用 DNA 折纸对神经元 trans-SNARE 复合物进行定量表征

• 批准号: 10281683
• 负责人: Zhao Zhang
• 金额: $ 41.43万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-15 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10281683
• 关键词: Affect; Basic Science; Binding; Cell membrane; Cleaved cell; Clostridial Neurotoxin; Complex; Consensus; DNA; DNA Structure; Disease; Docking; Electron Microscope; Environment; Exocytosis; Freezing; In Vitro; Individual; Investigation; Knowledge; Length; Libraries; Lipid Bilayers; Lipids; Measurement; Mediating; Membrane; Membrane Fusion; Membrane Proteins; Mental disorders; Methods; Molecular Conformation; Mutation; Nanotechnology; Neuraxis; Neurons; Neurosciences; Neurotoxicity Syndromes; Neurotoxins; Optics; Pathogenesis; Play; Positioning Attribute; Probability; Process; Proteins; Research; Resistance; Role; S-nitro-N-acetylpenicillamine; SNAP receptor; Spectrum Analysis; Structure; Synapses; Synaptic Cleft; Synaptic Receptors; Synaptic Vesicles; Techniques; Technology; Transmembrane Domain; Vesicle; Visualization; alpha helix; arm; base; clinical practice; density; driving force; experimental study; genetic regulatory protein; human disease; improved; interest; mechanical properties; mutant; nanodisk; nanoscale; neurophysiology; neurotransmission; neurotransmitter release; novel; prevent; programs; protein complex; protein function; reconstitution; recruit; scaffold; single molecule; stem; synaptotagmin I; syntaxin; tool; transmission process; vesicle-associated membrane protein

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.

项目摘要/摘要 神经传递的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中的裂口，在那里它们结合并激活后 突触受体。据信，一种称为SNARE的蛋白质络合物起着核心作用，因为它的组装可以 产生足够的能量来驱动融合。当前描述了SNARE介导的融合的假设是 称为“圈式拉链”：SV上的v-snare蛋白与pm上的T-nare蛋白异二聚体结合 形成跨弹络合物的拉链般的时尚（即V-和T-nare跨膜域是 嵌入单独的膜中）；释放的能量最终克服了排斥力 SV和PM并将两个膜拉在一起，在其中跨弹络合物转变为顺式sNare 复合物（即V-和T-Snares位于单个膜上）。目前大多数已知的内容 神经元素结构和动力学源于对顺式鼻子的分析，但“真正的英雄”跨环 这为膜融合提供了驱动力仍然难以捉摸。这里的主要技术挑战是 捕获在胞吞作用的快速过程（<1 ms）过程中部分组装的跨弹药配合物。 在此提案中，我们通过结合DNA的纳米级可编程性来提供解决方案 纳米技术和使用纳米轴（ND）限制融合孔扩展的能力。 V形DNA 折纸结构用于托管两个结合部分。一个部分由v-snares组成 在NDS中重构，而另一个则与同源T-Snares组成。我们的平台很大 通过研究来揭示神经元跨弹头组装的真实信息的改进方法：（1） 全长的圈圈蛋白而不是截断或突变，因为散发的破坏仅出现 从距离控制； （2）代表其本地环境的脂质双层中的小麻网。 在特定的目标1中，一组位于双层中的部分组装神经元跨鼻nare复合物是 产生，模仿突触融合机械中的渐进式四级核心。然后是各种梭质 神经毒素（CNT）被添加到复合体集合中，以及军鼓组装完整性之间的关系 并且可以系统地检查CNT的蛋白水解活性。在特定的目标2中，修改了V-origami 充当力光谱仪，研究神经元跨鼻子组件的能量景观 双层的背景。重要的是，我们将检查与疾病相关的编突变对 跨复合组装能量，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来重新审视中心而难以捉摸的机械之一 在神经科学中：神经元跨弹复合体。有关广泛使用的CNT和 预计与疾病相关的突变体将在这项研究中获取，并有可能受益于基础研究和 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。