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.

摘要 在拥挤的膜环境中，膜蛋白与其他可溶性和膜相关的 蛋白质和脂质形成大量的动态和瞬时蛋白质复合物， 细胞生理学也有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性质通常调节这些组装。 因此，为了了解特定膜蛋白之间的联系如何帮助细胞对一种免疫应答， 外部刺激，我们需要直接从蛋白质中研究相应蛋白质的低聚组装体。 脂双层环境。这就给我们带来了研究膜蛋白-脂质的主要挑战 交互.现有的研究这种相互作用的工具缺乏这种关键的能力， 直接从双层环境中进行分析。应对这一挑战， 该项目是开发一种新型实验平台，能够直接从内部分析MP复合物 体外脂质双层，其可定制为靶细胞膜。为此，我们将联合收割机 脂质囊泡技术与天然质谱（nativeMS）。在目标1中，取一组10个不同的 我们将开发一种实验方法，使我们能够 直接从一系列模拟不同生理学特性的脂质囊泡中确定它们的低聚状态， 膜。我们将验证和基准我们的结果对已知的低聚质量的每一个 这些蛋白质。这将建立我们的平台的适用性，以检测广泛的膜 来自各种脂质双层环境的蛋白质。在目标2中，我们将开发一种实验策略， 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。为此目的进行 我们与赛默飞世尔科技公司合作，将联合收割机ECD片段化与脂质囊泡nativeMS相结合 平台成功完成后，这两个目标将共同提供一个新的武器库， 技术研究膜蛋白和脂质的寡聚组织直接从一个 生理学相关的脂质双层。在目标3中，我们将把它应用到一个复杂的生物系统中， 神经生物学中的一个突出问题;充满神经递质的突触囊泡如何达到它们的超快速度 融合的速度为此，我们将专门针对突触囊泡突触素的作用 膜蛋白，它与各种神经系统疾病有关。所提出的实验可以 提出关键的机制和结构的见解，了解神经元信号转导和相关的 疾病特异性损伤。从长远来看，膜蛋白之间的联系受损， 与从神经变性到癌症的几种病理生理状况有关。我们 相信拟议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。