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

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.

摘要 大气中温室气体的含量，如甲烷，长期以来一直由微生物控制， 甲烷氧化菌最近的人类活动已经打破了这种稳态，对人类健康构成了明显的风险。 健康在现在和未来。颗粒甲烷单加氧酶（pMMO），一种铜依赖性 甲烷氧化细菌的跨膜酶，将甲烷氧化成甲醇。它执行这个任务的能力 在环境温度和压力下的困难的化学反应提供了一个窗口，以开发过程， 将生物天然气转化为液体（Bio-GTL），以缓解气候变化。pMMO的分离 膜和去污剂溶解阻碍了过去的研究，导致酶活性的损失， 蛋白质结构的扭曲。洗涤剂胶束不能概括洗涤剂的物理化学性质， 膜可以干扰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 交互.这些挑战可以通过在膜模拟物如膜中重构pMMO来克服。 支架蛋白（MSP）纳米盘（ND）和双胞，其使用均质合成脂质双层， pMMO活性和结构部分恢复。这个项目的目标是探索当地人的作用 pMMO结构和功能中的膜。目的1是优化pMMO在无洗涤剂天然ND中的活性 系统.初步数据表明，有可能使用天然脂质重建ND中的pMMO活性 从甲烷氧化菌中提取的这些天然脂质ND表现出与pMMO相当或更好的活性， 合成脂质ND。目的2是表征膜环境及其与pMMO的相互作用。 这些信息将用于优化膜模拟物，以描述脂质环境对细胞增殖的影响。 pMMO结构和功能。通过质谱法的非靶向和靶向脂质组学将用于分类 主要的脂质类，并确定具体的脂质种类，同时还确定其相对丰度， 天然脂质提取物和膜模拟物。天然质谱法将提供对特定蛋白质的深入了解- 膜模拟物内发生的脂质相互作用，为cryoEM中这些相互作用的建模提供信息 和晶体结构。目的3是表征膜模拟环境的结构效应 在pMMO。更天然样的膜模拟物可以允许确定更生物相关的生物学特性。 通过低温电子显微镜（cryoEM）观察pMMO结构。这些研究将提供深入了解 膜对pMMO功能的重要性，包括关于pMMO结构的关键细节，铜 中心，跨膜环，蛋白质-脂质相互作用，蛋白质-蛋白质相互作用，生理还原剂， 活性部位和机制。本项目还可提供关于 研究膜蛋白的天然膜环境。