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

SNARE蛋白和Sec 1 p/Munc 18家族（SM）蛋白构成了该基因的核心分子 介导几乎所有细胞内膜融合的机器。其他蛋白质调节 在正确的时间和地点进行核聚变这些功能障碍 蛋白质与神经和免疫紊乱、癌症、糖尿病 和其他疾病。几十年的研究已经证明，陷阱夫妇， 有序折叠和组装以SM蛋白依赖的方式进行膜融合。 然而，SM蛋白在SNARE组装中的作用机制尚不清楚， 以及SNARE组装如何与膜融合结合。使用高分辨率光学 镊子，我们最近发现，神经SM蛋白Munc 18 -1催化逐步 将三个突触SNARE（突触融合蛋白、VAMP 2和SNAP-25）组装成四螺旋 束，是神经传递和胰岛素分泌所必需的过程。重要的是， Munc 18 -1用作模板，以引导定向SNARE组装沿着新的 通路在本申请中，我们计划首先确定模板复合物是 SNARE-SM融合机制的保守中间体，以及其他 调节SNARE组装和膜融合的蛋白质。我们将研究 参与钙触发的胞吐作用的关键调节因子（Munc 13 -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， 同时检测逐步SNARE组装和状态式膜融合 大型纳米盘和被捕获的GUV。一个主要的目标是重建钙触发的 在受控的实验条件下进行膜融合，并了解其 工作机制最后，我们将扩展我们的方法，以确定分子 膜结合和脂质交换的机制，通过扩展的突触结合蛋白（E- Syts）和通过离子通道NOMPC的机械感觉。我们的长期目标是发展 阐明膜蛋白的稳定性、折叠和动力学的一般方法。

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.