Investigating protein‐membrane interactions using native reverse micelles constructed from naturally sourced lipids

Advancing the study of membrane associated proteins and their interactions is dependent on accurate membrane models. While a variety of membrane models for high‐resolution membrane protein study exist, most do not reflect the diversity of lipids found within biological membranes. In this work, we have developed native reverse micelles (nRMs) formulated with lipids from multiple eukaryotic sources, which encapsulate proteins and enable them to interact as they would with a biological membrane. Diverse formulations of nRMs using soy lecithin, porcine brain lipids, or bovine heart lipids combined with n‐dodecylphosphocholine were developed and characterized by dynamic light scattering and (31)P‐NMR. To optimize protein encapsulation, ubiquitin was used as a standard and protein NMR verified minimal changes to its structure. Peripheral membrane proteins, which bind reversibly to membranes, were encapsulated and include glutathione peroxidase 4 (GPx4), phosphatidylethanolamine‐binding protein 1 (PEBP1), and fatty acid binding protein 4 (FABP4). All three proteins showed anticipated interactions with the membrane‐like inner surface of the nRMs as assessed by protein NMR. The nRM formulations developed here allow for efficient, high‐resolution study of membrane interacting proteins up to and beyond ~21 kDa, in a more biologically relevant context compared to other non‐native membrane models. The approach outlined here may be applied to a wide range of lipid extracts, allowing study of a variety of membrane associated proteins in their specific biological context.