Native ion mobility mass spectrometry studies of potassium inward rectifier channels: insight into gating and lipid binding

项目概要/摘要 膜蛋白嵌入在生物膜中，在那里它们起作用并与生物膜密切相互作用。 脂质分子生物膜的环境是动态的，由丰富的化学物质组成 脂质分子的多样性。随着生物膜的复杂性，人们越来越认识到， 脂质分子在膜蛋白的折叠、结构和功能中的重要作用。特别是， 内向整流钾（Kir）通道在调节膜电位和钾离子通道中具有中心作用。 体内平衡近20年来，人们已经知道这些通道需要特定的脂质才能发挥作用。 尽管许多研究已经提供了对Kir通道结构和功能的深入了解，但仍然存在许多缺陷。 基本问题：是什么决定了Kir通道对脂质的选择性？有哪些 热力学结合参数的个别脂质结合事件的基尔通道？脂质会结合吗 与Kir频道合作Kir通道构象（开放或闭合）是否影响对 个体脂质？需要多少特定的脂质来控制Kir通道？什么是热力学 Kir通道开放和关闭状态的稳定性，当绑定到一个，两个，三个，或四个特定的脂质？这里我们 我建议使用本机离子迁移质谱（IM-MS）来解决这些基本问题 技术，从而在质谱仪中保留非共价相互作用，并利用IM-MS 与其他生物物理学方法不同，我们开创的方法使我们能够解决和询问 单个脂质结合事件与膜蛋白复合物。我们寻求应用新颖和高度创新的 IM-MS方法，我们最近开发的推导热力学结合参数的个人 膜蛋白的脂质结合事件。此外，我们建议使用IM-MS研究Kir通道门控 这将使我们第一次监测载脂蛋白和脂质结合的构象状态（开放和闭合）， states.我们的计划也将研究脂质和其他分子对Kir通道的变构调节 使用我们最近开发的新的IM-MS方法来解析和询问异质性脂质结合 单个脂质分辨率的事件。综上所述，我们预期我们提出的研究结果 提供脂质和其他分子如何调节Kir结构和功能的基本见解 渠道

Project Summary/Abstract Membrane proteins are embedded in the biological membrane where they function and intimately interact with lipid molecules. The environment of the biological membrane is dynamic and composed of a rich chemical diversity of lipid molecules. Alongside the complexity of the biological membrane is the growing realization of the important roles of lipid molecules in the folding, structure, and function of membrane proteins. In particular, inward rectifier potassium (Kir) channels have central roles in regulating membrane potential and potassium homeostasis. It has been known for nearly 20 years that these channels require a specific lipid for function. Although many studies have provided insight into Kir channel structure and function, there remain a number of fundamental questions: What determines the selectivity of Kir channels towards lipids? What are the thermodynamic binding parameters for individual lipid binding events to Kir channels? Do lipids bind cooperatively to Kir channels? Does Kir channel conformation (open or closed) influence the selectivity towards individual lipids? How many specific lipids are required to gate Kir channels? What are the thermodynamic stabilities of Kir channel open and closed states when bound to one, two, three, or four specific lipids? Here, we propose to address these fundamental questions using native ion mobility mass spectrometry (IM-MS) technology, whereby non-covalent interactions are preserved in the mass spectrometer and capitalize on IM-MS approaches we have pioneered that, unlike other biophysical methods, allow us to resolve and interrogate individual lipid binding events to membrane protein complexes. We seek to apply novel and highly innovative IM-MS approaches that we have recently developed to deduce thermodynamic binding parameters for individual lipid binding events to membrane proteins. Moreover, we propose to investigate Kir channel gating using IM-MS that will allow us for the first time to monitor the conformational states (open and closed) for apo and lipid bound states. Our proposal will also investigate the allosteric regulation of Kir channels by lipids and other molecules using new IM-MS methods we have recently developed to resolve and interrogate heterogeneous lipid binding events at the resolution of individual lipids. Taken together, we anticipate the results from our proposed studies to provide fundamental insight into how lipids and other molecules modulate the structure and function of Kir channels.