Study of the secondary structure of the C-terminal domain of the antiapoptotic protein Bcl-2 and its interaction with model membranes

Bcl-2 is a protein which inhibits programmed cell death. It is associated t o many cell membranes such as mitochondrial outer membrane, endoplasmic ret iculum, and nuclear envelope, apparently through a C-terminal hydrophobic d omain. We have used infrared spectroscopy to study the secondary structure of a synthetic peptide (a 23mer) with the same sequence as this C-terminal domain (residues 217-239) of Bcl-2. The spectrum of this peptide in D2O buf fer shows an amide I' band with a maximum at 1622 cm(-1), which clearly ind icates its tendency to aggregate in aqueous solvent. However, the peptide i ncorporated in multilamellar phosphatidylcholine membranes shows a totally different spectrum of the amide I' band, with a maximum at 1655 cm(-1), ind icating a predominantly ct-helical structure. Addition of the peptide to un ilamellar vesicles destabilized them and released encapsulated carboxyfluor escein. Differential scanning calorimetry of dimyristoylphosphatidylcholine multilamellar vesicles in which the peptide was incorporated revealed that increasing concentrations of the peptide progressively broadened the pretr ansition and the main transition, as is to be expected for a membrane integ ral molecule. Fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene in fluid phosphatidylcholine vesicles showed that increasing concentrations o f the peptide produced increased polarization values, pointing to an increa se in the apparent order of the membrane and indicating that high concentra tions of the peptide considerably broaden the phase transition of dimyristo ylphosphatidylcholine multilamellar vesicles. Quenching the intrinsic fluor escence of the Tyr-235 of the peptide, by KI, indicated that this aminoacyl residue is highly exposed to aqueous solvent when incorporated in phosphol ipid vesicles. The results are discussed in terms of their relevance to the proposed topology of insertion of Bcl-2 into biological membranes.