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

项目摘要/摘要 膜蛋白嵌入在生物膜中，它们在那里发挥作用并与之密切相互作用。 脂类分子。生物膜的环境是动态的，由丰富的化学物质组成 脂类分子的多样性。除了生物膜的复杂性之外，人们还日益认识到 脂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)通道在膜电位和钾离子的调节中起着中心作用 动态平衡。近20年来，人们已经知道这些通道需要一种特定的脂质才能发挥作用。 尽管许多研究已经为KIR通道的结构和功能提供了洞察力，但仍有许多 基本问题：是什么决定了KIR通道对脂质的选择性？什么是 个别脂质结合事件与KIR通道的热力学结合参数？脂类会绑定吗？ 合作到KIR频道？KIR通道构象(开放或关闭)是否影响对 单独的脂类？需要多少特定的脂类才能进入KIR通道？热力学是什么？ 当与一、二、三或四种特定脂类结合时，KIR通道开放和关闭状态的稳定性？在这里，我们 建议使用自然离子迁移质谱仪(IM-MS)来解决这些基本问题 技术，其中非共价相互作用被保存在质谱仪中并利用IM-MS 我们首创的方法，与其他生物物理方法不同，允许我们解决和审问 膜蛋白复合体的个别脂结合事件。我们寻求将新颖和高度创新的应用 我们最近开发的IM-MS方法，用于推导个体的热力学结合参数 膜蛋白的脂类结合事件。此外，我们还建议使用IM-MS来研究KIR信道选通 这将使我们第一次能够监测apo和脂质结合的构象状态(开放和关闭)。 各州。我们的计划还将研究脂类和其他分子对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相脂质结合 在个别脂质的分辨率下发生的事件。综上所述，我们期待着我们提议的研究的结果 提供对脂类和其他分子如何调节KIR结构和功能的基本见解 频道。

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.