Understanding organization of membrane proteins and lipids through lipid vesicle native mass spectrometry

摘要 在膜的拥挤环境中，膜蛋白以及其他可溶性和膜相关的 蛋白质和脂质形成大量动态和瞬时的蛋白质复合物，进而控制细胞 生理学。越来越多的证据表明，独立的膜蛋白-脂质相互作用以及 宿主膜的大量生物物理特性通常调节这些组件。因此，要了解如何 特定膜蛋白之间的关联有助于细胞对外部刺激做出反应，我们需要研究 直接来自脂质双层环境的各个蛋白质的寡聚组装体。这让我们 研究膜蛋白-脂质相互作用的主要挑战。研究此类问题的现有工具 相互作用缺乏直接从双层环境进行分子分析的关键能力。 为了应对这一挑战，该项目的总体目标是开发一个新颖的实验平台 能够直接从体外脂质双层分析 MP 复合物，可根据目标细胞进行定制 膜。为此，我们将脂质囊泡技术与天然质谱（nMS）相结合。瞄准 1、采用一组十种不同的标准寡聚膜蛋白我们将开发一种实验方法 这使我们能够直接从一系列模拟不同脂质囊泡的寡聚状态来确定它们的寡聚状态 生理膜。我们将根据已知的低聚物质量来验证和基准我们的结果 这些蛋白质中的每一个。这将建立我们的平台检测各种膜的适用性 来自各种脂质双层环境的蛋白质。在目标 2 中，我们将制定一项实验策略 使我们能够直接确定特异性结合的脂质结合以及它们结合的位置。为此，我们将 将 nMS 与基于脂质组学的 HPLC MS/MS 分析相结合，以确定脂质的身份。同时，在 与 Thermo Fisher Scientific 合作，我们将 ECD 片段化与脂质囊泡 nMS 平台结合起来 以确定脂质结合位点。成功完成后，这两个目标共同将提供 直接研究膜蛋白和脂质寡聚组织的新技术库 生理相关的脂质双层。在目标 3 中，我们将其应用于复杂的生物系统，以解决 神经生物学中的突出问题；充满神经递质的突触小泡如何达到超快的速度 融合。为此，我们将专门针对突触泡膜蛋白突触素的作用 这与各种神经系统疾病有关。所提出的实验可以得出关键的结论 机制和结构洞察力，以了解神经元信号转导和相关疾病特异性 损伤。从长远来看，膜蛋白之间的关联受损与多种因素有关。 病理生理状况范围从神经变性到癌症。我们对拟议的方案充满信心 该平台将在研究广泛的生物过程和相关疾病方面发挥变革性作用 州。

Abstract In the crowded milieu of the membrane, membrane proteins, with other soluble and membrane-associated proteins, and lipids form a large number of dynamic and transient protein complexes that in turn govern cellular physiology. There is mounting evidence that both independent membrane protein-lipid interactions, as well as bulk biophysical properties of the host membrane often regulate these assemblies. Hence, to understand how associations between specific membrane proteins help a cell responds to an external stimulus, we need to study the oligomeric assemblies of the respective proteins directly from the lipid bilayer environment. This brings us to the primary challenge of studying membrane protein-lipid interactions. The existing tools to study such interactions lack this critical ability to perform molecular analysis directly from the bilayer environment. Addressing this challenge, the overarching goal of this project is to develop a novel experimental platform that enables analysis of MP complexes directly from in vitro lipid bilayers, which can be customized to a target cellular membrane. To this end, we will combine lipid vesicle technologies with native mass spectrometry (nMS). In Aim 1, taking a set of ten different standard oligomeric membrane proteins we will develop an experimental method that enables us to determine their oligomeric states directly from a range of lipid vesicles mimicking different physiological membranes. We will validate and benchmark our results against the known oligomeric masses of each of these proteins. This will establish the applicability of our platform to detect a wide range of membrane proteins from a variety of lipid bilayer environments. In Aim 2, we will develop an experimental strategy that enables us to directly determine the specifically bound lipid binds and where do they bind. To this end, we will combine nMS with HPLC MS/MS analysis based lipidomics to determine the identity of the lipds. In parallel, in collaboration with Thermo Fisher Scientific, we will combine ECD fragmentation with lipid vesicle nMS platform to determine the site of lipid binding. Together, upon successful completion, these two Aims will provide an arsenal of new technologies to study the oligomeric organization of membrane proteins and lipids directly from a physiologically relevant lipid bilayer. In Aim 3, we will apply this to a complex biological system to address an outstanding question in neurobiology; how neurotransmitter filled synaptic vesicles attain their ultrafast speed of fusion. To this end, we will specifically target the role of synaptophysin, a synaptic vesicle membrane protein which has been linked to various neurological disorders. The experiment proposed can bring out critical mechanist and structural insight to understand neuronal signal transduction and related disease-specific impairments. In the long run, impairment of associations between membrane proteins has been linked to several pathophysiological conditions ranging from neurodegeneration to cancer. We are confident that the proposed platform will have a transformative role in studying a wide range of biological processes and associated disease states.