Engineered Nanodiscs for Structural Mass Spectrometry

项目摘要 该项目的总体目标是开发能够 制造具有详细和可调组合物的脂质纳米盘(NDS)，并利用这些 NDS作为改进的结构质谱学和蛋白质组学分析的载体 膜蛋白(MPS)。尽管在生物化学和 我们对MPS的了解远远落后于我们对MPS的了解 可溶性蛋白质及其功能复合体。这导致了严重的失衡，其中 MPS占药物靶标的60%以上，约占结构药物靶标的3%。 蛋白质数据库(PDB)中的条目。结果，MP靶向药物的发现变慢了，而且 治疗策略尚未被发现。 为了弥补这一差距，我们建议开发能够 在广泛的脂类成分上产生NDS，具有窄的粒度分布。我们 然后立即部署这些量身定做的NDS来研究 细胞色素P450(CYP)，一种存在于所有生命王国中的单调膜蛋白，以及 负责人类体内大多数小分子药物的新陈代谢。这是一种 环磷酰胺能够代谢如此广泛的外源物质，而细胞的作用 膜在细胞色素P450的表观结构可塑性中的作用，目前尚不清楚。我们会 发展离子迁移率-质谱仪(IM-MS)和碰撞诱导去折叠(CIU)方法 在我们的初步数据中，已经能够发现结构性转变的第一个证据 CYP作为其局部脂质环境的函数。我们将进一步努力，将NDS打造成 强大的提取设备，可提高膜蛋白质组的覆盖率。来补充 除了我们的IM-MS工作流程外，我们还将部署和优化化学交联(CXL) 与NDS内的MPS一起使用的方法。 上面讨论的工具将被应用于研究MP络合物在 线粒体膜。具体地说，我们将针对与以下相关的蛋白质组件 电子传输链(ETC)，以及大量的蛋白质-蛋白质相互作用 (PPI)，已经观察到或预测到了CYP。我们的工作将寻求提供新的 关于过去被广泛研究的络合物的结构信息(例如，ATP 合酶)，以及寻求发现新的线粒体MP复合体，具有潜在的 对细胞功能的广泛影响。

Project Summary The overall objective of this project is to develop new microfluidic devices capable of producing lipid nanodiscs (NDs) having detailed and tunable compositions, and utilize these NDs as vehicles for improved structural mass spectrometry (SMS) and proteomics assays of membrane proteins (MPs). Despite positions of prominence in both biochemistry and the pharmaceutical sciences, our understanding of MPs lags significantly behind our knowledge of soluble proteins and their functional complexes. This has led to a critical imbalance, where MPs, which account for greater than 60% of drug targets, account for ~3% of the structural entries in the protein data bank (PDB). As a result, MP-targeted drug discovery is slowed, and treatment strategies go undiscovered. To bridge this gap, we propose the development of monolithic microfluidic tools capable of producing NDs over a wide range of lipid compositions, having narrow size distributions. We will then immediately deploy these tailored NDs to study the structure and biophysics of cytochrome P450 (CYP), a monotopic membrane protein found in all kingdoms of life, and responsible for the metabolism of most small molecule drugs in humans. The means by which CYPs are able to metabolize such a wide range of xenobiotics, and the role that cellular membranes play in the apparent structural plasticity of CYPs, remains unknown. We will develop ion mobility-mass spectrometry (IM-MS) and collision induced unfolding (CIU) methods that, in our preliminary data, have been able to detect the first evidence for structural shifts in CYP as a function of its local lipid environment. Our efforts will further extend to build NDs into robust extraction devices for improved coverage of the membrane proteome. To complement our IM-MS workflows, we will also deploy and optimize chemical cross-linking (CXL) approaches for use with MPs housed within NDs. The tools discussed above will then be applied to the study of the MP complexes within the mitochondrial membrane. Specifically, we will target the protein assemblies associated with the electron transport chain (ETC), as well as the vast array of protein-protein interactions (PPIs) that have either been observed or predicted for CYP. Our work will seek to provide new structural information on complexes that have been studied extensively in the past (e.g. ATP Synthases), as well as seek to discover new mitochondrial MP complexes, with potentially broad implications for cellular function.