Reconstitution of the Melibiose Permease of Salmonella enterica serovar Typhimurium (MelBSt) into Lipid Nanodiscs.

Abstract

Membrane proteins play critical roles in cell physiology and pathology. The conventional way to study membrane proteins at protein levels is to use optimal detergents to extract proteins from membranes. Identification of the optimal detergent is tedious , and in some cases, the protein functions are compromised. While this detergent-based approach has produced meaningful results in membrane protein research, a lipid environment should be more suitable to recapture the protein's native folding and functions. This protocol describes how to prepare amphipathic membrane scaffold-proteins (MSPs)-based nanodiscs of a cation-coupled melibiose symporter of Salmonella enterica serovar Typhimurium (MelBSt), a member of the major facilitator superfamily. MSPs generate nano-assemblies containing membrane proteins surrounded by a patch of native lipids to better preserve their native conformations and functions. This protocol requires purified membrane protein in detergents, purified MSPs in solution, and detergent-destabilized phospholipids. The mixture of all three components at specific ratios is incubated in the presence of Bio-Beads SM-2 resins, which absorb all detergent molecules, allowing the membrane protein to associate with lipids surrounded by the MSPs. By reconstituting the purified membrane proteins back into their native-like lipid environment, these nanodisc-like particles can be directly used in cryo-EM single-particle analysis for structure determination and other biophysical analyses. It is noted that nanodiscs may potentially limit the dynamics of membrane proteins due to suboptimal nanodisc size compared to the native lipid bilayer. Key features • This protocol was built based on the method originally developed by Sligar et al. [1] and modified for a specific major facilitator superfamily transporter • This protocol is robust and reproducible • Lipid nanodiscs can increase membrane protein stability, and reconstituted transporters in lipid nanodiscs can regain function if their function is compromised using detergents • The reconstituted lipids nanodisc can be used for cryo-EM single-particle analysis.