# Investigating the role of the membrane in particulate methane monooxygenase (pMMO) structure and function > **NIH NIH F31** · NORTHWESTERN UNIVERSITY · 2024 · $37,131 ## Abstract 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. ## Key facts - **NIH application ID:** 10893369 - **Project number:** 5F31ES034283-03 - **Recipient organization:** NORTHWESTERN UNIVERSITY - **Principal Investigator:** Frank Tucci - **Activity code:** F31 (R01, R21, SBIR, etc.) - **Funding institute:** NIH - **Fiscal year:** 2024 - **Award amount:** $37,131 - **Award type:** 5 - **Project period:** 2022-09-01 → 2025-05-31 ## Primary source NIH RePORTER: https://reporter.nih.gov/project-details/10893369 ## Citation > US National Institutes of Health, RePORTER application 10893369, Investigating the role of the membrane in particulate methane monooxygenase (pMMO) structure and function (5F31ES034283-03). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10893369. Licensed CC0. --- *[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*