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

摘要 SNARE蛋白和Sec1p/Munc18家族(SM)蛋白构成了 介导几乎所有的细胞膜融合。其他蛋白质调节核心机制以 在正确的时间和地点启用融合。这些蛋白质的功能障碍与 神经和免疫紊乱、癌症、糖尿病和其他疾病。几十年来 研究证实，圈套将它们有序的折叠和组装连接到膜上 以SM蛋白依赖的方式融合。然而，目前还不清楚SM扮演的是什么机械性角色 蛋白质在SNARE组装中的作用，以及SNARE组装如何耦合到膜融合。vbl.使用 高分辨率光钳，我们最近发现神经元SM蛋白Munc18-1催化 将三个突触圈套(Synaxin、VAMP2和SNAP-25)逐步组装成一个四螺旋 束，这是神经传递和胰岛素分泌必不可少的过程。重要的是，Munc18-1 用作模板以引导定向诱捕组件沿新路径行进。在此应用程序中， 我们计划首先确定模板复合体是SNARE-SM融合的保守中间体 也是其他蛋白质调节圈套组装和膜融合的关键靶点。 我们将研究参与钙触发胞吐的关键调节因子的作用(Munc13-1， 络合素、突触素、神经生长因子和α-SNAP)与SNARE和SM蛋白的磷酸化 关于陷阱的组装和拆卸。然后，我们将开发新的检测方法来同时检测 利用大纳米盘和捕获器的步进式陷阱组装和状态膜融合 古斯。一个主要的目标是在受控下重建钙触发的膜融合 并了解它们的工作机理。最后，我们将延长我们的 确定膜结合和脂质交换分子机制的方法学 扩展突触素(E-Syts)和离子通道NOMPC的机械感觉。我们的长期合作 目的是开发一种通用的方法来阐明膜的稳定性、折叠和动力学。 蛋白质。

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.