Production of membrane-embedded Bcl-2 proteins - Use of cell-free synthesis in continuous exchange for co-translational insertion of Bcl-2 proteins in lipid bilayer nanodiscs

The BCL-2 family proteins are key regulators of apoptosis, functionally divided in pro- and anti-apoptotic proteins, with a third group acting as regulators. Their ability to partition between the cytosol and intra-cellular membranes (essentially the mitochondrial outer membrane) is a primary regulator of their functions. A second contributor is their ability to form homotypic complexes (pro-pro or anti-anti) or heterotypic complexes (pro-anti). If the structures of monomeric cytosolic members have largely been characterized, the functional and structural study of membrane-embedded proteins remains incomplete. Unlocking this knowledge is expected to enable evaluating new therapeutic strategies to either activate pro-apoptotic members, or inactivate anti-apoptotic ones. Lipid bilayer nanodiscs and improved cell-free protein synthesis have provided the technical breakthrough to achieve the description at the atomic level of conformations and higher order assemblies of these proteins in their membrane-associated states. Here we describe detailed and straightforward protocols for generating nanodisc-inserted members of the Bcl-2 family, through the example of anti-apoptotic Bcl-xL, and pro-apoptotic Bax and Bak. Full-length, untagged proteins are expressed from bacterial extracts in the presence of pre-assembled nanodiscs to allow co/post-translational insertion in lipid bilayer, followed by affinity chromatography purification. A more detailed characterization is presented for Bak, to exemplify structural and mechanistic studies enabled by these methods. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/745005v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@5da1d1org.highwire.dtl.DTLVardef@12aca96org.highwire.dtl.DTLVardef@5a3e73org.highwire.dtl.DTLVardef@ba009d_HPS_FORMAT_FIGEXP M_FIG C_FIG