Reconstitution of mitochondrial membrane proteins into nanodiscs by cell-free expression

Abstract

The isolation and characterization of mitochondrial membrane proteins is technically challenging because they natively reside within the specialized environment of the lipid bilayer, an environment that must be recapitulated to some degree during reconstitution to ensure proper folding, stability, and function. Here we describe protocols for the assembly of a membrane protein into lipid bilayer nanodiscs in a series of cell-free reactions. Cell-free expression of membrane proteins circumvents problems attendant with in vivo expression such as cytotoxicity, low expression levels, and the formation of inclusion bodies. Nanodiscs are artificial membrane systems comprised of discoidal lipid bilayer particles bound by annuli of amphipathic scaffold protein that shield lipid acyl chains from water. They are therefore excellent platforms for membrane protein reconstitution and downstream solution-based biochemical and biophysical analysis. This chapter details the procedures for the reconstitution of a mitochondrial membrane protein into nanodiscs using two different types of approaches: cotranslational and posttranslational assembly. These strategies are broadly applicable for different mitochondrial membrane proteins. They are also applicable for the use of nanodiscs with distinct lipid compositions that are biomimetic for different mitochondrial membranes and that recapitulate lipid profiles associated with pathological disorders in lipid metabolism.