Unravelling Membrane Protein-Lipid Interactions using Nanodiscs and Mass Spectrometry

项目总结/摘要 膜蛋白参与许多细胞过程，因此是广泛应用的关键药物靶标。 疾病。然而，在了解全球如何改变脂质方面存在根本性的差距， 环境影响局部膜蛋白的结构和功能。越来越多的证据表明， 是膜蛋白功能所必需的，但很难确定其分子机制 蛋白质-脂质相互作用主要的挑战是，传统的结构生物学工具和结合测定 不太适合表征瞬时和异质蛋白质-脂质相互作用。 为了增进我们对生物过程的理解，为推进疾病治疗奠定基础， 我们的目标是开发新的方法来确定脂双层如何调节膜蛋白。研究一 从细菌复合物到病毒离子通道，再到人类， 运输商，我们专注于回答几个关键问题。 首先，哪些脂质结合给定的膜蛋白靶点？膜蛋白中经常观察到脂质 结构，但确定局部脂质组周围存在的脂质的身份可能具有挑战性 膜蛋白我们将使用新的脂质组学脂质交换-质谱法来研究富集 含有膜蛋白靶点的纳米盘脂蛋白颗粒中的特定脂质种类。我们的目标是 在天然脂质的复杂混合物中鉴定结合膜蛋白靶的未知脂质。 第二，脂质是如何与蛋白质相互作用的？我们知道脂质对于细胞膜的形成 蛋白质的功能，但往往不清楚在哪里以及如何具体结合。我们将开发新的本地质量 光谱分析方法来确定脂质与膜蛋白靶标结合的位点和选择性。我们 目标是揭示在特定结合位点驱动脂质特异性的分子机制。 最后，为什么脂质对膜蛋白功能很重要？我们知道双层细胞脂质 调节疾病，年龄和环境，但目前还不清楚这些全球脂质的变化 影响局部膜蛋白生理学。我们将研究膜蛋白靶点在不同细胞中的功能， 脂质环境和影响脂质结合的不同突变体。对于显著影响 功能，我们将进行结构分析，以连接脂质结合在特定网站与功能的结果。 我们的首要目标是了解全球脂质组学变化如何影响局部膜蛋白结构 和功能这将通过识别对维持蛋白质活性重要的脂质来影响生物医学研究 并有助于阐明天然双层内膜蛋白的生理机制。最后， 对蛋白质-脂质相互作用的更好的理解具有改进药物发现和 通过调节细胞脂质来调节膜蛋白活性的新治疗策略。

PROJECT SUMMARY/ABSTRACT Membrane proteins are involved in many cellular processes and thus are critical drug targets for a wide range of diseases. However, there is a fundamental gap in understanding how the global changes to the lipid environment affect local membrane protein structure and function. Mounting evidence indicates that lipids can be essential for membrane protein function, but it is difficult to determine the molecular mechanisms underlying protein-lipid interactions. The primary challenge is that conventional structural biology tools and binding assays are poorly suited to characterizing transient and heterogeneous protein-lipid interactions. To advance our understanding of biological process and lay a foundation for advancing disease treatment, our goal is to develop new approaches to determine how lipid bilayers regulate membrane proteins. Studying a diverse set of membrane protein targets ranging from bacterial complexes to viral ion channels to human transporters, we are focused on answering several key questions. First, which lipids bind a given membrane protein target? Lipids are often observed in membrane protein structures, but it can be challenging to determine the identity of the lipids present in the local lipidome surrounding membrane proteins. We will use novel lipidomic lipid exchange-mass spectrometry methods to study enrichment of specific lipid species in nanodisc lipoprotein particles containing the membrane protein target. Our goal is to identify unknown lipids that bind the membrane protein targets in complex mixtures of natural lipids. Second, how and were do lipids interact with the protein? We know that lipids can be critical for membrane protein function, but it is often unclear where and how specifically they bind. We will develop new native mass spectrometry methods to determine the sites and selectivity of lipid binding to membrane protein targets. Our goal is to uncover the molecular mechanisms driving lipid specificity at specific binding sites. Finally, why are lipids important for membrane protein function? We know that bunk cellular lipids are modulated in response to disease, age, and the environment, but it is unclear how these global lipid changes affect local membrane protein physiology. We will study the function of membrane protein targets in different lipid environments and with different mutants that affect lipid binding. For lipid sites that significantly affect function, we will perform structural analysis to connect lipid binding at specific sites with functional outcomes. Our overarching goal is to understand how global lipidomic changes affect local membrane protein structure and function. This will impact biomedical research by identifying lipids important for maintaining protein activity and aiding in elucidating the physiological mechanisms of membrane proteins inside natural bilayers. Ultimately, an improved understanding of protein-lipid interactions holds the potential for improved drug discovery and for new therapeutic strategies for modulating membrane protein activity by modulating cellular lipids.