Single-molecule manipulation of proteins involved in membrane fusion, lipid exchange, and mechanosensation

摘要 SNARE蛋白和Sec1p/Munc18家族（SM）蛋白构成核心分子机器， 介导几乎所有的细胞内膜融合。其他蛋白质调节核心机制， 在正确的时间和地点进行核聚变。这些蛋白质的功能障碍与 神经和免疫紊乱、癌症、糖尿病和其它疾病。几十年的 研究已经证实，SNARE将它们的有序折叠和组装耦合到膜上， 以SM蛋白依赖的方式融合。然而，目前尚不清楚SM的机制作用是什么。 蛋白质在SNARE组装中的作用以及SNARE组装如何与膜融合偶联。使用 高分辨率光镊，我们最近发现，神经元SM蛋白Munc18 - 1催化 三个突触SNARE（突触融合蛋白、VAMP 2和SNAP-25）逐步组装成四螺旋 束，这是神经传递和胰岛素分泌所必需的过程。重要的是，Munc18 - 1 用作引导定向SNARE组装沿着新路径的模板。在本申请中， 我们计划首先确定模板复合物是SNARE-SM融合的保守中间体 这些蛋白质是调节SNARE组装和膜融合的关键靶点。 我们将研究参与钙触发的胞吐作用的关键调节因子的作用（Munc13 - 1， 复合蛋白、突触结合蛋白、NSF和α-SNAP）以及SNARE和SM蛋白的磷酸化 关于SNARE组装和拆卸。然后，我们将开发新的检测方法， 使用大纳米盘和捕获的逐步SNARE组装和状态式膜融合 GUV。一个主要的目标是重建钙触发的膜融合在控制下， 实验条件，并了解其工作机理。最后，我们将扩大我们的 方法来查明膜结合和脂质交换的分子机制， 延伸的突触结合蛋白（E-Syts）和通过离子通道NOMPC的机械感觉。我们的长期 目的是发展一种通用的方法来阐明膜的稳定性，折叠和动力学 proteins.

Abstract SNARE proteins and Sec1p/Munc18-family (SM) proteins constitute the core molecular machines that mediate nearly all intracellular membrane fusion. Other proteins regulate the core machinery to enable fusion at the right time and location. Dysfunction of these proteins has been linked to neurological and immunological disorders, cancers, diabetes, and other diseases. Decades of research have established that SNAREs couple their ordered folding and assembly to membrane fusion in a SM protein-dependent manner. However, it remains unclear what mechanistic role SM proteins plays in SNARE assembly and how SNARE assembly is coupled to membrane fusion. Using high-resolution optical tweezers, we recently found that neuronal SM protein Munc18-1 catalyzes step-wise assembly of three synaptic SNAREs (syntaxin, VAMP2, and SNAP-25) into a four-helix bundle, a process essential for neurotransmission and insulin secretion. Importantly, Munc18-1 serves as a template to guide directional SNARE assembly along a new pathway. In this application, we plan to first establish that the template complex is a conserved intermediate for SNARE-SM fusion machineries and a key target for other proteins to regulate SNARE assembly and membrane fusion. We will examine effects of key regulators involved in calcium-triggered exocytosis (Munc13-1, complexin, synaptotagmin, NSF, and alpha-SNAP) and phosphorylation of SNARE and SM proteins on SNARE assembly and disassembly. Then, we will develop new assays to simultaneous detect step-wise SNARE assembly and state-wise membrane fusion using large nanodiscs and trapped GUVs. A major goal is to reconstitute the calcium-triggered membrane fusion under controlled experimental conditions and to understand their working mechanism. Finally, we will extend our methodologies to pinpoint the molecular mechanisms of membrane binding and lipid exchange by extended synaptotagmins (E-Syts) and of mechanosensation by ion channel NOMPC. Our long-term goal is to develop a general approach to elucidate stability, folding, and dynamics of membrane proteins.