Characterization of the membrane-inserted C-terminus of cytoprotective BCL-XL.

BCL-XL is a dominant inhibitor of apoptosis and a significant anti-cancer drug target. Endogenous BCL-XL is integral to the mitochondrial outer membrane (MOM). BCL-XL reconstituted in detergent-free lipid bilayer nanodiscs is anchored to the nanodisc lipid bilayer membrane by tight association of its C-terminal tail, while the N-terminal head retains the canonical structure determined for water-soluble, tail-truncated BCL-XL, with the surface groove solvent-exposed and available for BH3 ligand binding. To better understand the conformation and dynamics of this key region of BCL-XL we have developed methods for isolating the membrane-embedded C-terminal tail from its N-terminal head and for preparing protein suitable for structural and biochemical studies. Here, we outline the methods for sample preparation and characterization and describe previously unreported structural and dynamics features. We show that the C-terminal tail of BCL-XL forms a transmembrane α-helix that retains a significant degree of conformational dynamics. We also show that the presence of the intact C-terminus destabilizes the soluble state of the protein, and that the small fraction of soluble recombinant protein produced in Escherichia coli is susceptible to proteolytic degradation of C-terminal residues beyond M218. This finding impacts the numerous previous studies where recombinant soluble BCL-XL was presumed to be full-length. Nevertheless, the majority of recombinant BCL-XL produced in E. coli is insoluble and protected from proteolysis. This protein retains the complete C-terminal tail and can be reconstituted in lipid bilayers in a folded and active state.