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，在理解脂质环境如何影响膜的方面存在根本性的差距。 蛋白质结构与功能越来越多的证据表明，脂质可能是膜蛋白的必需物质， 功能，但它是具有挑战性的，以确定蛋白质的重要性，脂质的分子机制 交互.主要的挑战是，传统的结构生物学工具和结合测定很差， 适用于表征瞬时和异质蛋白质-脂质相互作用。 为了了解脂质如何调节膜蛋白的结构和功能，我的研究计划是 开发新的工具，通过结合脂蛋白纳米盘和质量来研究蛋白质-脂质相互作用 质谱法（MS）。这些新技术将使用特征良好的细菌膜开发 蛋白质复合物，然后应用于更复杂的哺乳动物蛋白质，如视紫红质和解偶联 蛋白质2.我们的目标是回答给定膜蛋白靶点的四个问题。1)脂质与 目标？为了鉴定目标周围的内源性脂质，我们正在开发一种混合物， 脂肽/脂蛋白方法将由其天然脂质包围的膜溶解成纳米盘 而不需要洗涤剂。在目标纯化之后，我们将提取和鉴定 自然与目标有关。2)脂质与目标结合的强度如何？区分紧密结合的 从弱相关脂质中分离脂质，我们将用不同的脂质混合物组装脂蛋白纳米盘， 使用本机MS对完整的纳米盘组装件进行扫描。使用碰撞激活，我们将逐渐分离 纳米圆盘测量脂质环带和紧密结合的结构脂质的组成。 此外，我们将在实验中使用纳米盘之间的脂质交换来测量脂质结合常数 类似于平衡透析。3)脂质在哪里与蛋白质结构相互作用？在预测血脂- 结合位点，我们将通过突变相互作用的残基来测试预测， 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 重要功能？通过将MD、诱变和天然MS与功能研究配对，我们将连接 对蛋白质功能的脂质依赖性影响，具有特异性脂质结合位点。 我们的首要目标是开发一个工具箱，用于解开蛋白质-脂质的分子机制 交互.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理解具有改进药物发现的潜力， 膜蛋白靶点和用于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.