Biophys J, January 2002, p. 233-243, Vol. 82, No. 1

The Structure of the C-Terminal Domain of the Pro-Apoptotic Protein Bak and Its Interaction with Model Membranes

Departamento de Bioquímica y Biología Molecular-A, Edificio de Veterinaria, Universidad de Murcia, E-30100 Murcia, Spain

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-¹⁸⁸ILNVLVVLGVVLLGQFVVRRFFKS²¹¹-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 cm¹, which clearly indicates the predominance of an extended -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 cm¹, indicating a predominantly -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 -helical structure and considerably perturbs the physical properties of the membrane.

Biophys J, January 2002, p. 233-243, Vol. 82, No. 1
© 2002 by the Biophysical Society   0006-3495/02/01/233/11  $2.00

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