Bcl-2 is a protein which inhibits programmed cell death. It is associated to many cell membranes such as mitochondrial outer membrane, endoplasmic reticulum, and nuclear envelope, apparently through a C-terminal hydrophobic domain. 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 D(2)O buffer shows an amide I' band with a maximum at 1622 cm(-1), which clearly indicates its tendency to aggregate in aqueous solvent. However, the peptide incorporated in multilamellar phosphatidylcholine membranes shows a totally different spectrum of the amide I' band, with a maximum at 1655 cm(-)(1), indicating a predominantly alpha-helical structure. Addition of the peptide to unilamellar vesicles destabilized them and released encapsulated carboxyfluorescein. Differential scanning calorimetry of dimyristoylphosphatidylcholine multilamellar vesicles in which the peptide was incorporated revealed that increasing concentrations of the peptide progressively broadened the pretransition and the main transition, as is to be expected for a membrane integral molecule. Fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene in fluid phosphatidylcholine vesicles showed that increasing concentrations of the peptide produced increased polarization values, pointing to an increase in the apparent order of the membrane and indicating that high concentrations of the peptide considerably broaden the phase transition of dimyristoylphosphatidylcholine multilamellar vesicles. Quenching the intrinsic fluorescence of the Tyr-235 of the peptide, by KI, indicated that this aminoacyl residue is highly exposed to aqueous solvent when incorporated in phospholipid vesicles. The results are discussed in terms of their relevance to the proposed topology of insertion of Bcl-2 into biological membranes.
Calorimetry, Differential Scanning Diphenylhexatriene