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

通过脂质囊泡天然质谱了解膜蛋白和脂质的组织

• 批准号: 10612847
• 负责人: Kallol Gupta
• 金额: $ 35.18万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-01 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10612847
• 关键词: Ablation; Accounting; Address; Benchmarking; Binding; Binding Sites; Biochemical; Biological; Biological Assay; Biological Models; Biological Process; Cell membrane; Cell physiology; Cells; Cellular Membrane; Chemicals; Collaborations; Complex; Crowding; Data; Detection; Detergents; Disease; Endoplasmic Reticulum; Environment; Goals; Human; Impairment; In Vitro; Lateral; Link; Lipid Bilayers; Lipid Binding; Lipids; Liquid substance; Malignant Neoplasms; Mass Spectrum Analysis; Membrane; Membrane Lipids; Membrane Proteins; Methodology; Methods; Mitochondria; Molecular; Molecular Analysis; Mutation; Nerve Degeneration; Neurobiology; Neurons; Neurotransmitters; Pathologic; Physiological; Play; Property; Proteins; Proteome; Protocols documentation; Role; SNAP receptor; Signal Pathway; Signal Transduction; Speed; Stimulus; Synapses; Synaptic Vesicles; Synaptophysin; System; Technology; Vesicle; biophysical properties; cell growth; cell growth regulation; cellular targeting; complex biological systems; detection platform; drug market; experimental study; insight; membrane assembly; membrane reconstitution; nervous system disorder; neurotransmission; neurotransmitter release; new technology; next generation; novel; pressure; protein complex; reconstitution; stoichiometry; technological innovation; tool; vesicular release

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 also 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 (nativeMS). 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, in collaboration with Thermo Fisher Scientific, we will combine ECD fragmentation with lipid vesicle nativeMS platform. 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.

抽象的 在膜的拥挤环境中，膜蛋白与其他可溶性和膜相关 蛋白质和脂质形成大量动态和瞬态蛋白质复合物，而蛋白质又决定 细胞生理。也有越来越多的证据表明两种独立的膜蛋白脂质 宿主膜的相互作用以及块状生物物理特性通常调节这些组件。 因此，要了解特定膜蛋白之间的关联如何帮助细胞响应 外部刺激，我们需要直接研究各个蛋白的寡聚组件 脂质双层环境。这使我们面临研究膜蛋白脂质的主要挑战 互动。研究这种相互作用的现有工具缺乏执行分子的关键能力 直接从双层环境分析。解决这个挑战，这是总体目标 项目是开发一个新型的实验平台，该平台可以直接从IN中对MP复合物进行分析 体外脂质双层，可以定制为靶细胞膜。为此，我们将结合 具有天然质谱法（Nativems）的脂质囊泡技术。在AIM 1中，取十个不同的 标准的寡聚膜蛋白我们将开发一种实验方法，使我们能够 直接从模仿不同生理的一系列脂质囊泡中确定其低聚物状态 膜。我们将针对每个已知的低聚质量验证和基准测试我们的结果 这些蛋白质。这将确定我们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AIM 2中，我们将制定一种实验策略， 使我们能够直接确定特异性结合的脂质结合以及它们在哪里结合。为此， 与Thermo Fisher Scientific的合作，我们将ECD碎片与脂质囊泡Nativems相结合 平台。成功完成后，这两个目标将提供新的库 直接从A的技术研究膜蛋白和脂质的寡聚组织 生理上相关的脂质双层。在AIM 3中，我们将将其应用于复杂的生物系统以解决 神经生物学方面的杰出问题；神经递质填充的突触囊泡如何达到其超快 融合速度。为此，我们将特别靶向突触囊泡突触素的作用 膜蛋白与各种神经系统疾病有关。提出的实验可以 提出关键的机械师和结构见解，以了解神经元信号转导和相关 疾病特异性障碍。从长远来看，膜蛋白之间关联的损害具有 与几种从神经变性到癌症的病理生理状况有关。我们是 确信所提出的平台将在研究广泛的生物学方面发挥变革性作用 过程和相关疾病状态。