Unravelling Membrane Protein-Lipid Interactions using Nanodiscs and Mass Spectrometry

项目摘要/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点。 疾病的威胁。然而，在理解全球范围内脂质如何变化方面存在着根本的差距 环境影响局部膜蛋白的结构和功能。越来越多的证据表明，脂类可以 对于膜蛋白的功能是必不可少的，但很难确定其潜在的分子机制 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分析 不太适合描述瞬时和异相的蛋白质-脂相互作用。 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质双层如何调节膜蛋白。学习a 多种膜蛋白靶标，从细菌复合体到病毒离子通道再到人类 运输商们，我们现在专注于回答几个关键问题。 首先，哪些脂类结合了特定的膜蛋白靶标？脂类常见于膜蛋白中。 结构，但要确定存在于局部脂体周围的脂类的身份可能是具有挑战性的 膜蛋白。我们将使用新的脂类脂质交换-质谱学方法来研究富集物 含有膜蛋白靶标的纳米盘脂蛋白颗粒中的特定类脂物种。我们的目标是 在天然脂类的复杂混合物中识别结合膜蛋白靶标的未知脂类。 第二，脂类如何以及如何与蛋白质相互作用？我们知道脂类对细胞膜至关重要。 蛋白质的功能，但通常不清楚它们在哪里以及如何具体结合。我们将发展新的本土群众 光谱分析方法确定脂类与膜蛋白靶标结合的位置和选择性。我们的 目的是揭示在特定结合部位驱动脂质专一性的分子机制。 最后，为什么脂类对膜蛋白的功能很重要？我们知道双层细胞脂类是 根据疾病、年龄和环境进行调节，但尚不清楚这些全球脂质是如何变化的 影响局部膜蛋白生理。我们将研究膜蛋白靶标在不同环境中的功能。 脂质环境和影响脂质结合的不同突变体。对于显著影响 对于功能，我们将进行结构分析，将特定部位的脂质结合与功能结果联系起来。 我们的首要目标是了解全球脂肪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类来影响生物医学研究 有助于阐明天然双层内膜蛋白的生理机制。最终， 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.