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，在理解脂质环境如何影响细胞膜方面存在着根本的差距。 蛋白质结构和功能。越来越多的证据表明，脂类对膜蛋白是必不可少的。 功能，但要确定蛋白质-脂肪重要性的分子机制是具有挑战性的 互动。主要的挑战是传统的结构生物学工具和结合分析很差。 适用于描述瞬时和非均相蛋白质-脂质相互作用。 为了了解脂类如何调节膜蛋白的结构和功能，我的研究项目是 利用脂蛋白纳米盘与质量相结合开发研究蛋白质-脂质相互作用的新工具 光谱(MS)。这些新技术将使用具有良好特性的细菌膜进行开发 蛋白质复合体在应用于更复杂的哺乳动物蛋白质之前，如视紫红质和解偶联 我们的目标是针对给定的膜蛋白靶标回答四个问题。1)脂类与哪些物质相互作用 目标是什么？为了确定靶子周围的内源性脂质，我们正在开发一种混合体 脂肽/脂蛋白方法将天然脂类包裹的膜溶解成纳米盘 不需要洗涤剂。在纯化靶标后，我们将提取并鉴定 自然地与目标联系在一起。2)脂质与靶标结合的强度有多大？区分紧紧捆绑的 脂类，我们将用不同的脂类和脂类的混合物组装脂蛋白纳米盘。 使用原生MS对完整的纳米盘组件进行电离。利用碰撞激活，我们将逐渐解离 该纳米盘用于测量脂质环带和紧密相关的结构脂的组成。 此外，我们将在一项实验中使用纳米盘之间的脂交换来测量脂结合常数 类似于平衡透析。3)脂类在哪里与蛋白质结构相互作用？在预测了血脂之后- 结合位置使用分子动力学(MD)，我们将通过突变相互作用残基和 使用天然MS检测结合位点的破坏。4)为什么特定的蛋白质-脂质相互作用 对功能重要吗？通过将MD、突变和天然多发性硬化与功能研究配对，我们将把 具有特定脂结合部位的脂依赖对蛋白质功能的影响。 我们的首要目标是开发一个工具箱，用于解开蛋白质-脂肪的分子机制。 互动。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的潜力 膜蛋白靶标和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 lipid environment affects membrane protein structure and function. Mounting evidence indicates that lipids can be essential for membrane protein function, but it is challenging to determine the molecular mechanisms underlying the importance of 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 understand how lipids modulate membrane protein structure and function, my research program is developing new tools to study protein-lipid interactions by combining lipoprotein nanodiscs with mass spectrometry (MS). These new technologies will be developed using well-characterized bacterial membrane protein complexes before being applied to more complex mammalian proteins such as rhodopsin and uncoupling protein 2. Our goal is to answer four questions for a given membrane protein target. 1) What lipids interact with the target? To identify the endogenous lipids that surround the target, we are developing a hybrid lipopeptide/lipoprotein approach to solubilize membranes surrounded by their natural lipids into nanodiscs without the need for detergent. Following purification of the target, we will extract and identify the lipids that are naturally associated with the target. 2) How strongly do lipids bind to the target? To distinguish tightly bound lipids from weakly associated lipids, we will assemble lipoprotein nanodiscs with different mixtures of lipids and use native MS to ionize the intact nanodisc assembly. Using collisional activation, we will gradually dissociate the nanodisc to measure the composition of the lipid annular belt and tightly associated structural lipids. Furthermore, we will use lipid exchange between nanodiscs to measure lipid binding constants in an experiment analogous to equilibrium dialysis. 3) Where do lipids interact with the protein structure? After predicting lipid- binding sites using molecular dynamics (MD), we will test the predictions by mutating interacting residues and using native MS to detect the disruption of the binding sites. 4) Why are specific protein-lipid interactions important for function? By pairing MD, mutagenesis, and native MS with functional studies, we will connect lipid-dependent effects on protein function with specific lipid binding sites. Our overarching goal is to develop a toolbox for unravelling the molecular mechanisms of protein-lipid interactions. 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 with membrane protein targets and for new therapeutic strategies for modulating membrane protein activity.