CONTRIBUTION OF THE HYDROPHOBICITY GRADIENT OF AN AMPHIPATHIC PEPTIDETO ITS MODE OF ASSOCIATION WITH LIPIDS

A class of peptides that associate with lipids, known as oblique-orien tated peptides, was recently described [Brasseur R., Pillet, T., Lins, L., Vandekerckhove, J. & Rosseneu, M. (1997) Trends Biochem. Sci. 22, 167-171]. Due to an asymmetric distribution of hydrophobic residues a long the axis of the alpha-helix, such peptides adopt an oblique orien tation which can destabilise membranes or lipid cores. Variants of the se oblique peptides, designed to have an homogeneous distribution of h ydrophobic and hydrophilic residues along the helical axis, are classi fied as regular amphipathic peptides. These peptides are expected to l ie parallel to the polar/apolar interface with their hydrophobic resid ues directed towards the apolar and their hydrophilic residues towards the polar phase. An hydrophobic, oblique-orientated peptide was ident ified at residues 56-68 in the sequence of the lecithin-cholesterol ac yltransferase (LCAT), enzyme. This peptide is predicted to penetrate a lipid bilayer at an angle of 40 degrees through its more hydrophobic C-terminal end and thereby induce the destabilisation of a membrane or a lipid core. The LCAT-(56-68) wild-type peptide was synthesised toge ther with the LCAT-(56-68, 0 degrees) variant, in which the hydrophobi city gradient was abolished through residue permutations. In two other variants, designed to keep their oblique orientation, the W61 residue was shifted either towards the more hydrophilic N-terminal at residue 57, or to position 68 at the hydrophobic C-terminal end of the peptid e. Peptide-induced vesicle fusion was demonstrated by fluorescence mea surements using pyrene-labeled vesicles and by monitoring of vesicle s ize by gel filtration. The interaction between peptides and lipids was monitored by measurement of the intrinsic tryptophan fluorescence emi ssion of the peptides. Fluorescence polarisation measurements, using d iphenyl hexatriene, were carried out to follow changes in the lipid fl uidity. The LCAT-(56-68) wild-type peptide and the two oblique variant s, induced fusion of unilamellar dimyristoylglycerophosphocholine vesi cles. Tryptophan fluorescence emission measurements showed a 12-14 nm blue shift upon addition of the wild-type peptide and of the W61-->68 variant to lipids, whereas the fluorescence of the W61-->57 variant di d not change significantly. This observation supports the insertion of the more hydrophobic C-terminal residues into the lipid phase, as pre dicted by the theoretical calculations. In contrast, the 0 degrees var iant peptide had no fusogenic activity, and it associated with lipids to form small discoidal lipid/peptide complexes. The phospholipid tran sition temperature was decreased after addition of the wild-type, the W61-->68 and W61-->57 fusogenic peptides, whereas the opposite effect was observed with the 0 degrees variant. The behaviour of the wild-typ e and variant LCAT-(56-68) peptides stresses the contribution of the h ydrophobicity gradient along the axis of an amphipathic peptide to the mode of association of this peptide with lipids. This parameter conse quently influences the structural modifications occurring to lipids up on association with amphipathic peptides.