Engineered Nanodiscs for Structural Mass Spectrometry

用于结构质谱分析的工程纳米圆盘

• 批准号: 10460573
• 负责人: Philip C Andrews
• 金额: $ 34.12万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-22 至 2024-07-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10460573
• 关键词: Adrenodoxin; Area; Binding; Biochemistry; Biological; Biological Assay; Biological Process; Biophysics; Cell physiology; Cells; Cellular Membrane; Cellular biology; Chemicals; Code; Complement; Complex; Coupled; Cytochrome P450; Cytochromes; Data; Development; Devices; Drug Targeting; Electron Transport; Elements; Engineering; Environment; Evaluation; Genes; Goals; Human; Knowledge; Lead; Libraries; Life; Lipid Binding; Lipids; Mass Spectrum Analysis; Membrane; Membrane Proteins; Metabolism; Methods; Microfluidic Microchips; Microfluidics; Miniaturization; Mitochondrial Membrane Protein; Mitochondrial Proteins; Mixed Function Oxygenases; Molecular Conformation; NADPH-Ferrihemoprotein Reductase; Peptide Signal Sequences; Pharmaceutical Preparations; Pharmacologic Substance; Phosphorylation; Physiological; Play; Positioning Attribute; Preparation; Protein Array; Protein Dynamics; Proteins; Proteome; Proteomics; Protocols documentation; Role; Science; Structure; System; Technology; Time; Transmembrane Domain; Ursidae Family; Work; Xenobiotics; base; crosslink; drug discovery; drug metabolism; experimental study; human disease; improved; inhibitor; ion mobility; mitochondrial membrane; nanodisk; protein complex; protein data bank; protein function; protein protein interaction; protein structure; protein structure function; small molecule; structural biology; tool; treatment strategy

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.

项目摘要 该项目的总体目的是开发能够的新的微流体设备 生产具有详细和可调成分的脂质纳米盘（NDS），并利用这些脂质 ND作为改进结构质谱法（SMS）和蛋白质组学测定法的车辆 膜蛋白（MPS）。尽管生物化学和 药学科学，我们对国会议员的理解大大落后于我们的知识 可溶性蛋白及其功能复合物。这导致了严重的失衡，在这里 国会议员占药物靶标的60％以上的国会议员，占结构的约3％ 蛋白质数据库（PDB）中的条目。结果，以MP为目标的药物发现减慢，并且 治疗策略未被发现。 为了弥合这一差距 在各种脂质组合物上产生ND，具有狭窄的分布。我们 然后，将立即部署这些量身定制的ND，以研究 细胞色素P450（CYP），一种在生活的所有王国中发现的单位膜蛋白， 负责人类中大多数小分子药物的代谢。用的手段 CYP能够代谢如此广泛的异种生物，以及细胞的作用 膜在CYP的明显结构可塑性中发挥作用，仍然未知。我们将 开发离子迁移率质谱（IM-MS）和碰撞诱导的展开方法（CIU）方法 在我们的初步数据中，这已经能够检测到结构性转移的第一个证据 CYP是其本地脂质环境的函数。我们的努力将进一步扩展到建立ND 可靠的提取装置可改善膜蛋白质组的覆盖率。补充 我们的IM-MS工作流程，我们还将部署和优化化学交联（CXL） 与NDS内包含的国会议员一起使用的方法。 然后，上面讨论的工具将应用于对内部MP复合物的研究 线粒体膜。具体而言，我们将针对与之相关的蛋白质组件 电子传输链（ETC）以及大量的蛋白质蛋白质相互作用 （PPI）已被观察或预测CYP。我们的工作将寻求提供新的 过去对复合物进行了广泛研究的结构信息（例如ATP 合成酶），并试图发现新的线粒体MP复合物，并具有潜在的 对细胞功能的广泛意义。