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.

项目概要 该项目的总体目标是开发能够 生产具有详细且可调节成分的脂质纳米盘（ND），并利用这些 ND 作为改进结构质谱 (SMS) 和蛋白质组学分析的载体 膜蛋白（MP）。尽管在生物化学和生物化学领域都具有突出的地位 在制药科学领域，我们对 MP 的理解远远落后于我们对药物科学的了解 可溶性蛋白质及其功能复合物。这导致了严重的不平衡，其中 MP 占药物靶点的 60% 以上，约占结构性靶点的 3% 蛋白质数据库（PDB）中的条目。结果，MP 靶向药物的发现速度减慢，并且 治疗策略未被发现。 为了弥补这一差距，我们建议开发能够 生产范围广泛的脂质成分的ND，具有狭窄的尺寸分布。我们 然后将立即部署这些定制的 ND 来研究 细胞色素 P450 (CYP)，一种存在于所有生命王国中的单位膜蛋白，以及 负责人体大多数小分子药物的代谢。所采取的手段 CYP 能够代谢如此广泛的异生物质，并且细胞的作用 膜在 CYP 的表观结构可塑性中发挥的作用仍然未知。我们将 开发离子淌度质谱 (IM-MS) 和碰撞诱导解折叠 (CIU) 方法 在我们的初步数据中，我们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。我们的努力将进一步延伸，将 ND 建设成为 强大的提取装置可提高膜蛋白质组的覆盖率。来补充 我们的 IM-MS 工作流程中，我们还将部署和优化化学交联 (CXL) 与 ND 内的 MP 一起使用的方法。 然后，上述工具将应用于 MP 复合物的研究 线粒体膜。具体来说，我们将针对与相关的蛋白质组装体 电子传递链 (ETC)，以及大量的蛋白质-蛋白质相互作用 (PPIs) 已观察到或预测的 CYP。我们的工作将寻求提供新的 过去已被广泛研究的复合物的结构信息（例如 ATP Synthases），并寻求发现新的线粒体 MP 复合物，具有潜在的用途 对细胞功能的广泛影响。