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

摘要 在拥挤的膜环境中，膜蛋白与其他可溶性和膜相关 蛋白质和脂类形成大量动态的和瞬时的蛋白质复合体，这些复合体反过来又支配着 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整体生物物理性质经常调节这些组装。 因此，要了解特定膜蛋白之间的关联如何帮助细胞对 外部刺激，我们需要研究各自蛋白质的寡聚组装直接从 脂质双层环境。这给我们带来了研究膜蛋白-脂质的主要挑战 互动。现有的研究这种相互作用的工具缺乏这种关键的能力来执行分子 直接从双层环境进行分析。应对这一挑战，这一行动的首要目标是 项目是开发一个新的实验平台，能够直接从In 体外脂质双层，可定制为目标细胞膜。为此，我们将联合 脂泡技术与天然质谱学(NativeMS)。在目标1中，选择一组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状态的脂泡中确定它们的低聚状态 膜。我们将验证我们的结果，并将其与每个已知的寡聚体质量进行比较 这些蛋白质。这将建立我们的平台的适用性，以检测广泛的薄膜 来自各种脂类双层环境的蛋白质。在目标2中，我们将开发一种实验性策略， 使我们能够直接确定特定结合的脂类结合以及它们结合的位置。为此，在 与Thermo Fisher Science合作，我们将结合ECD碎裂和脂泡NativeMS 站台。成功完成后，这两个目标将共同提供一个新的武器库 直接研究膜蛋白和膜脂低聚体组织的技术 生理上相关的脂质双分子层。在目标3中，我们将把这一点应用于一个复杂的生物系统来解决 神经生物学中的一个悬而未决的问题：充满神经递质的突触小泡如何达到超快 聚变的速度。为此，我们将特别针对突触素的作用，突触小泡 一种膜蛋白，与多种神经疾病有关。提出的实验可以 提出关键的机械师和结构洞察力来理解神经元信号转导和相关 疾病特有的损伤。从长远来看，膜蛋白之间联系的损害 与多种病理生理疾病有关，从神经退化到癌症。我们是 相信拟议的平台将在研究广泛的生物学领域发挥变革性的作用 进程和相关的疾病状态。

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.