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，如 作为甲烷氧化菌。最近的人类活动破坏了这种动态平衡，给人类带来了明显的风险。 现在和未来的健康。颗粒甲烷单加氧酶(PMMO)，一种铜依赖的 来自甲烷氧化细菌的跨膜酶，将甲烷氧化成甲醇。它有能力做到这一点 在常温常压下的复杂化学反应提供了一扇开发工艺的窗口 将生物天然气转化为液体(Bio-GTL)以缓解气候变化。从PMMO中分离pMMO 膜和洗涤剂的增溶阻碍了过去的研究，导致酶活性和 蛋白质结构的扭曲。洗涤剂胶束未能概括洗涤剂的物理化学性质 膜可能扰乱具有重要功能的金属中心、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类似膜的膜模拟物中重组pMMO来克服 支架蛋白(MSP)纳米盘(NDS)和使用均一合成脂质双层的双分子束，这使得 PMMO活性和结构部分恢复。这个项目的目标是探索当地人的角色 膜在pMMO中的结构和功能。目标1是优化无洗涤剂天然ND中的pMMO活性 系统。初步数据显示，使用天然脂质重建NDS中的pMMO活性是可能的 从甲烷氧化菌中提取。这些天然脂质NDS的活性与pMMO相当或更好 合成脂NDS。目的2是表征膜环境及其与pMMO的相互作用。 这些信息将被用来优化膜模拟，以描绘脂环境对 PMMO的结构和功能。通过质谱学的非靶向和靶向脂质组学将被用于分类 主要的脂类，并鉴定特定的脂类，同时还测定它们在 天然脂类提取物和膜模拟物。天然质谱学将提供对特定蛋白质的洞察- 在膜模拟物中发生的脂类相互作用，为低温电子显微镜中这些相互作用的建模提供信息 和晶体结构。目标3是表征膜模拟环境的结构效应 在pMMO上。更像天然的膜模拟物可能允许确定更具生物学相关性的 低温电子显微镜(CryoEM)观察pMMO结构。这些研究将为我们提供对 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-脂相互作用，蛋白质-蛋白质相互作用，生理还原剂， 主动位置，主动机制。该项目还可以提供关于 研究膜蛋白的天然膜环境。