Martin Lidman

Reconstitution of the anti-apoptotic Bcl-2 protein into lipid membranes and biophysical evidence for its detergent-driven association with the pro-apoptotic Bax protein.

The anti-apoptotic B-cell CLL/lymphoma-2 (Bcl-2) protein and its counterpart, the pro-apoptotic Bcl-2-associated X protein (Bax), are key players in the regulation of the mitochondrial pathway of apoptosis. However, how they interact at the mitochondrial outer membrane (MOM) and there determine whether the cell will live or be sentenced to death remains unknown. Competing models have been presented that describe how Bcl-2 inhibits the cell-killing activity of Bax, which is common in treatment-resistant tumors where Bcl-2 is overexpressed. Some studies suggest that Bcl-2 binds directly to and sequesters Bax, while others suggest an indirect process whereby Bcl-2 blocks BH3-only proteins and prevents them from activating Bax. Here we present the results of a biophysical study in which we investigated the putative interaction of solubilized full-length human Bcl-2 with Bax and the scope for incorporating the former into a native-like lipid environment. Far-UV circular dichroism (CD) spectroscopy was used to detect direct Bcl-2-Bax-interactions in the presence of polyoxyethylene-(23)-lauryl-ether (Brij-35) detergent at a level below its critical micelle concentration (CMC). Additional surface plasmon resonance (SPR) measurements confirmed this observation and revealed a high affinity between the Bax and Bcl-2 proteins. Upon formation of this protein-protein complex, Bax also prevented the binding of antimycin A2 (a known inhibitory ligand of Bcl-2) to the Bcl-2 protein, as fluorescence spectroscopy experiments showed. In addition, Bcl-2 was able to form mixed micelles with Triton X-100 solubilized neutral phospholipids in the presence of high concentrations of Brij-35 (above its CMC). Following detergent removal, the integral membrane protein was found to have been fully reconstituted into a native-like membrane environment, as confirmed by ultracentrifugation and subsequent SDS-PAGE experiments.

CHAPTER 7:Membranes and Their Lipids: A Molecular Insight into Their Organization and Function

Lipids are a highly diverse family of amphiphilic molecules that form the basis of biological membranes. These are fascinating molecules that assemble spontaneously into thin membrane leaflets without which life would not have evolved. Membranes possess unique physicochemical properties and functions, which cannot be understood solely on the individual lipid level but is ultimately linked to their unique 2D assembly into a membrane. To understand the structural and dynamic organization of membranes, ranging from the atomic level to a specific lipid and finally to the macroscopic lipid assembly, solid-state NMR in combination with high-resolution-like magic angle spinning (MAS) techniques has been invaluable over the last two decades. Here, we focus on solid-state MAS NMR of natural abundance nuclei, such as 31P, 14N, 13C and even 1H, as specific reporters that provide information about the hydrophilic membrane exterior and hydrophobic membrane core. We focus on three areas of membrane research: (i) the tracking of individual lipids in living/intact systems and obtaining their lipid profiles (lipidomics) in the hunt for promising biomarkers; (ii) the impact of individual lipids, namely oxidized phospholipids, on membrane organization; and (iii) the role of these lipids in apoptosis by regulating protein function at the mitochondrial membrane level.