Membrane-Mimicking Reverse Micelles for High-Resolution Interfacial Study of Proteins and Membranes

Despite substantial advances, the study of proteins interacting with membranes remains a significant challenge. While integral membrane proteins have been a major focus of recent efforts, peripheral membrane proteins (PMPs) and their interactions with membranes and lipids have far less high-resolution information available. Their small size and the dynamic nature of their interactions have stalled detailed interfacial study using structural methods like cryo-EM and X-ray crystallography. A major roadblock for the structural analysis of PMP interactions is limitations in membrane models to study the membrane recruited state. Commonly used membrane mimics such as liposomes, bicelles, nanodiscs, and micelles are either very large or composed of non-biological detergents, limiting their utility for the NMR study of PMPs. While there have been previous successes with integral and peripheral membrane proteins, currently employed reverse micelle (RM) compositions are optimized for their inertness with proteins rather than their ability to mimic membranes. Applying more native, membrane-like lipids and surfactants promises to be a valuable advancement for the study of interfacial interactions between proteins and membranes. Here, we describe the development of phosphocholine-based RM systems that mimic biological membranes and are compatible with high-resolution protein NMR. We demonstrate new formulations that are able to encapsulate the model soluble protein, ubiquitin, with minimal perturbations of the protein structure. Furthermore, one formula, DLPC:DPC, allowed the encapsulation of the PMPs glutathione peroxidase 4 (GPx4) and phosphatidylethanolamine-binding protein 1 (PEBP1) and enabled the embedment of these proteins, matching the expected interactions with biological membranes. Dynamic light scattering and small-angle X-ray scattering characterization of the RMs reveals small, approximately spherical, and non-aggregated particles, a prerequisite for protein NMR and other avenues of study. The formulations presented here represent a new tool for the study of elusive PMP interactions and other membrane interfacial investigations.