Contribution of the hydrophobicity gradient of an amphipathic peptide to its mode of association with lipids.
A class of peptides that associate with lipids, known as oblique-orientated peptides, was recently described [Brasseur R., Pillot, T., Lins, L., Vandekerckhove, J. & Rosseneu, M. (1997) Trends Biochem. Sci. 22, 167-171]. Due to an asymmetric distribution of hydrophobic residues along the axis of the alpha-helix, such peptides adopt an oblique orientation which can destabilise membranes or lipid cores. Variants of these oblique peptides, designed to have an homogeneous distribution of hydrophobic and hydrophilic residues along the helical axis, are classified as regular amphipathic peptides. These peptides are expected to lie parallel to the polar/apolar interface with their hydrophobic residues directed towards the apolar and their hydrophilic residues towards the polar phase. An hydrophobic, oblique-orientated peptide was identified at residues 56-68 in the sequence of the lecithin-cholesterol acyltransferase (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 together with the LCAT-(56-68, 0 degrees) variant, in which the hydrophobicity 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 peptide. Peptide-induced vesicle fusion was demonstrated by fluorescence measurements using pyrene-labeled vesicles and by monitoring of vesicle size by gel filtration. The interaction between peptides and lipids was monitored by measurement of the intrinsic tryptophan fluorescence emission of the peptides. Fluorescence polarisation measurements, using diphenyl hexatriene, were carried out to follow changes in the lipid fluidity. The LCAT-(56-68) wild-type peptide and the two oblique variants, induced fusion of unilamellar dimyristoylglycerophosphocholine vesicles. 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 did not change significantly. This observation supports the insertion of the more hydrophobic C-terminal residues into the lipid phase, as predicted by the theoretical calculations. In contrast, the 0 degrees variant peptide had no fusogenic activity, and it associated with lipids to form small discoidal lipid/peptide complexes. The phospholipid transition 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-type and variant LCAT-(56-68) peptides stresses the contribution of the hydrophobicity gradient along the axis of an amphipathic peptide to the mode of association of this peptide with lipids. This parameter consequently influences the structural modifications occurring to lipids upon association with amphipathic peptides.
Laboratory for Lipoprotein Chemistry, Universiteit Gent, Belgium.
O
O
Pérez-Méndez
B
B
Vanloo
A
A
Decout
M
M
Goethals
F
F
Peelman
J
J
Vandekerckhove
R
R
Brasseur
M
M
Rosseneu
D020134:Catalytic Domain; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003198:Computer Simulation; D004789:Enzyme Activation; D006860:Hydrogen Bonding; D050356:Lipid Metabolism; D008055:Lipids / Q000737:chemistry; D008958:Models, Molecular; D009391:Nephelometry and Turbidimetry; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D007862:Phosphatidylcholine-Sterol O-Acyltransferase / Q000737:chemistry / Q000378:metabolism; D014867:Water
D008055:Lipids; D010446:Peptide Fragments; D010455:Peptides; D014867:Water; D007862:Phosphatidylcholine-Sterol O-Acyltransferase