Bax C-Terminal Peptide - Insights Into Membrane Interactions

Multi-cellular organisms eliminate unnecessary or defective cells through a process known as apoptosis. This tightly regulated series of events, leading to the concise shutting down and packaging up of the cellular machinery, is aptly coined “programmed cell death”. The core apoptotic machinery is composed of members of the Bcl-2 family of proteins. Each protein has a specific yet apparently redundant function, as single pro-apoptotic protein deletions do not render the whole system non-functional. The members act synergistically as initiators, effectors and antagonists of apoptosis. The commitment to self-destruct hinges on Bax, the proverbial “final straw”, facilitating the abrupt release of mitochondrial matrix proteins, setting off an irreversible avalanche of biochemical events including proteolysis and nuclear fragmentation. Though a wealth of data exists on the apoptotic process in general and the Bcl2 family in particular, the precise mechanism by which Bax interacts with and disrupts the structural integrity of the mitochondrial membrane remains elusive. Structural studies infer that the pro-apoptotic function of Bax is mediated by the insertion of the C-terminal helix as well as helices α5-α6 into the mitochondrial membrane. Conversely, the NMR structure of monomeric Bax clearly shows the hydrophobic α5-α6 helices are completely sequestered within the protein; furthermore, the putative regulatory domain is constrained by the presence of the C-terminal helix tucked solidly into its hydrophobic groove, implicating the need for a major conformational change for those interactions to occur. In order to clarify the mechanism by which Bax interacts with the mitochondrial membrane, we have measured the binding affinity, orientation, and depth of insertion of synthetic peptides, corresponding to the last 25 residues of the Bax C-terminus, to artificial mitochondrial membranes by circular dichroism (CD), resonance energy transfer (RET), fluorescence quenching and attenuated total reflectance-fourier transform infrared spectroscopy (ATR-FTIR).