The structure of the C-terminal domain of the pro-apoptotic protein bak and its interaction with model membranes

Bak is a pro-apoptotic protein widely distributed in different cell types that is associated with the mitochondrial outer membrane, apparently through a C-terminal hydrophobic domain. We used infrared spectroscopy to study the secondary structure of a synthetic peptide (+3HN-(188)ILNVLVVLGVVLLGQFVVRRFFKS(211)-COO-) with the same sequence as the C-terminal domain of Bak. The spectrum of this peptide in D2O buffer shows an amide I' band with a maximum at 1636 cm(-1), which clearly indicates the predominance of an extended beta-structure in aqueous solvent. However, the peptide incorporated in multilamellar dimyristoylphosphatidylcholine (DMPC) membranes shows a different amide I' band spectrum, with a maximum at 1658 cm(-1), indicating a predominantly alpha-helical structure induced by its interaction with the membrane. It was observed that through differential scanning calorimetry the transition of the phospholipid model membrane was broadened in the presence of the peptide. Fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) in fluid DMPC vesicles showed that increasing concentrations of the peptide produced increased polarization values, which is compatible with the peptide being inserted into the membrane. High concentrations of the peptide considerably broaden the phase transition of DMPC multilamellar vesicles, and DPH polarization increased, especially at temperatures above the T-c transition temperature of the pure phospholipid. The addition of peptide destabilized unilamellar vesicles and released encapsulated carboxyfluorescein. These results indicate that this domain is able to insert itself into membranes, where it adopts an alpha-helical structure and considerably perturbs the physical properties of the membrane.