Investigating the role of the membrane in particulate methane monooxygenase (pMMO) structure and function

研究膜在颗粒甲烷单加氧酶 (pMMO) 结构和功能中的作用

• 批准号: 10676098
• 负责人: Frank Tucci
• 金额: $ 4.29万
• 依托单位: NORTHWESTERN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10676098
• 关键词: Active Sites; Address; Affect; Atmosphere; Bacteria; Bathing; Biochemical; Biological; Biophysics; Catalogs; Chemistry; Consumption; Copper; Cryoelectron Microscopy; Data; Detergents; Electron Transport; Environment; Environmental Health; Enzymes; Equation; Exhibits; Failure; Future; Goals; Health; Homeostasis; Human; Human Activities; Hydrophobicity; Interdisciplinary Study; Knowledge; Lipid Bilayers; Lipids; Liquid substance; Mass Spectrum Analysis; Membrane; Membrane Proteins; Metals; Methane; Methane Metabolism Pathway; Methane hydroxylase; Methanol; Methylococcaceae; Methylococcus capsulatus; Micelles; Microbe; Modeling; Molecular Structure; Natural Gas; Outcome; Oxidoreductase; Particulate; Pathway interactions; Physiological; Play; Process; Property; Proteins; Recovery; Reducing Agents; Research; Resolution; Risk; Role; Route; Scaffolding Protein; Scientist; Structure; System; Temperature; Testing; Training Programs; Transmembrane Domain; Visualization; anthropogenesis; atmospheric carbon dioxide; chemical reaction; climate change; climate instability; design; future pandemic; greenhouse gases; innovation; insight; interest; lipidomics; mimetics; nanodisk; oxidation; partial recovery; preservation; pressure; protein protein interaction; protein structure; reconstitution; tropospheric ozone; Active Sites; Address; Affect; Atmosphere; Bacteria; Bathing; Biochemical; Biological; Biophysics; Catalogs; Chemistry; Consumption; Copper; Cryoelectron Microscopy; Data; Detergents; Electron Transport; Environment; Environmental Health; Enzymes; Equation; Exhibits; Failure; Future; Goals; Health; Homeostasis; Human; Human Activities; Hydrophobicity; Interdisciplinary Study; Knowledge; Lipid Bilayers; Lipids; Liquid substance; Mass Spectrum Analysis; Membrane; Membrane Proteins; Metals; Methane; Methane Metabolism Pathway; Methane hydroxylase; Methanol; Methylococcaceae; Methylococcus capsulatus; Micelles; Microbe; Modeling; Molecular Structure; Natural Gas; Outcome; Oxidoreductase; Particulate; Pathway interactions; Physiological; Play; Process; Property; Proteins; Recovery; Reducing Agents; Research; Resolution; Risk; Role; Route; Scaffolding Protein; Scientist; Structure; System; Temperature; Testing; Training Programs; Transmembrane Domain; Visualization; anthropogenesis; atmospheric carbon dioxide; chemical reaction; climate change; climate instability; design; future pandemic; greenhouse gases; innovation; insight; interest; lipidomics; mimetics; nanodisk; oxidation; partial recovery; preservation; pressure; protein protein interaction; protein structure; reconstitution; tropospheric ozone

ABSTRACT The atmospheric content of greenhouse gases, such as methane, has long been ruled by microbes, such as methanotrophs. Recent human activity has upset this homeostasis, presenting an appreciable risk to human health in the present and future. Particulate methane monooxygenase (pMMO), a copper-dependent transmembrane enzyme from methanotrophic bacteria, oxidizes methane to methanol. Its ability to perform this difficult chemical reaction at ambient temperature and pressure offers a window into developing processes for conversion of biological natural gas to liquid (Bio-GTL) for climate change mitigation. Isolation of pMMO from the membranes and detergent solubilization have hindered past studies, resulting in a loss of enzymatic activity and distortion of protein structure. The failure of detergent micelles to recapitulate the physicochemical properties of the membrane may perturb functionally important metal centers, protein-lipid interactions, and protein-protein interactions. These challenges can be overcome by reconstituting pMMO in membrane mimetics like membrane scaffold protein (MSP) nanodiscs (NDs) and bicelles using homogeneous synthetic lipid bilayers, which enable partial recovery of pMMO activity and structure. The goal of this project is to explore the role of the native membrane in pMMO structure and function. Aim 1 is to optimize pMMO activity in detergent-free native ND systems. Preliminary data show that it is possible to reconstitute pMMO activity in NDs using native lipids extracted from methanotrophs. These native lipid NDs exhibit activity comparable to or better than pMMO in synthetic lipid NDs. Aim 2 is to characterize the membrane environment and its interaction with pMMO. This information will be used to optimize membrane mimetics for delineating the effects of lipid environment on pMMO structure and function. Untargeted and targeted lipidomics via mass spectrometry will be used to catalog the major lipid classes and identify specific lipid species, while also determining their relative abundances in native lipid extracts and membrane mimetics. Native mass spectrometry will provide insight into specific protein- lipid interactions that occur within membrane mimetics, informing the modeling of these interactions in cryoEM and crystal structures. Aim 3 is to characterize the structural effects of membrane mimetic environments on pMMO. More native-like membrane mimetics may allow for determination of a more biologically relevant pMMO structure by cryogenic electron microscopy (cryoEM). These studies will provide insight into the importance of the membrane for pMMO function, including crucial details about the pMMO structure, copper centers, transmembrane loops, protein-lipid interactions, protein-protein interactions, physiological reductant, active site, and mechanism. This project may also provide generalizable information about the importance of the native membrane environment for studying membrane proteins.

抽象的 温室气体的大气含量，例如甲烷，长期以来一直由微生物统治，例如 作为甲烷营养。最近的人类活动使这种体内平衡感到不安，对人类带来了明显的风险 现在和未来的健康。颗粒甲烷单氧酶（PMMO），铜依赖性 来自甲嗜营养细菌的跨膜酶将甲烷氧化为甲醇。它有能力执行此功能 在环境温度和压力下的难以化学反应为开发过程提供了一个窗口 将生物天然气转换为液体（Bio-GTL），以缓解气候变化。隔离PMMO从 膜和洗涤剂溶解化已阻碍了过去的研究，导致酶活性丧失和 蛋白质结构的变形。洗涤剂胶束未能概括的物理化学特性 膜可能会扰动功能上重要的金属中心，蛋白质脂质相互作用和蛋白质 - 蛋白质 互动。这些挑战可以通过在膜（如膜）中重新建立PMMO来克服 使用均匀的合成脂质双层的脚手架蛋白（MSP）纳米散发（NDS）和双丝 PMMO活性和结构的部分恢复。该项目的目的是探索本地人的作用 PMMO结构和功能中的膜。 AIM 1是优化无污染的天然ND中的PMMO活性 系统。初步数据表明，可以使用本机脂质在NDS中重新建立PMMO活动 从甲烷营养中提取。这些天然脂质ND的活性与PMMO相当或更好 合成脂质NDS。目的2是表征膜环境及其与PMMO的相互作用。 此信息将用于优化膜模拟物，以描绘脂质环境对 PMMO结构和功能。通过质谱法的未靶向和靶向脂质组学将用于分类 主要的脂质类并确定特定的脂质物种，同时也确定其相对丰度 天然脂质提取物和膜模拟物。天然质谱法将洞悉特定蛋白质 - 在膜模拟物中发生的脂质相互作用，告知Cryoem这些相互作用的建模 和晶体结构。目标3是表征膜模拟环境的结构效应 在PMMO上。更类似天然的膜模拟物可能可以确定更具生物学相关的 低温电子显微镜（冷冻）的PMMO结构。这些研究将为您提供有关 膜对于PMMO功能的重要性，包括有关PMMO结构，铜的关键细节 中心，跨膜环，蛋白 - 脂质相互作用，蛋白质 - 蛋白质相互作用，生理还原剂， 主动部位和机制。该项目还可以提供有关该项目的重要信息 用于研究膜蛋白的天然膜环境。