Association of synthetic peptide fragments of human apolipoprotein A-I with phospholipids.

The sequences of the plasma apolipoproteins have a high degree of internal homology as they contain several 22-mer internal repeats. These amphipathic helical repeats are considered as the structural and functional units of this class of proteins. We proposed that the 22-mer repeats of the plasma apolipoproteins consist of 17-mer helical segments separated by extended beta-strands comprising five amino acid residues with a proline in the center of this segment. These beta-strand segments help reverse the orientation of the consecutive helices of apoA-I, A-IV, and E in a discoidal apolipoprotein-phospholipid complex. In order to support this hypothesis, we synthesized apoA-I fragments consisting of, respectively, one putative helix (residues 166-183), one helix plus a beta-strand (residues 161-183), and a pair of helices separated by a beta-strand (residues 145-183). The structural and lipid-binding properties of these peptides were investigated by turbidity, fluorescence, binding studies with unilamellar phospholipid vesicles, electron microscopy, and circular dichroism measurements. Our data show that one single putative helical segment or one helical segment plus one extended beta-strand do not form stable complexes with phospholipids. The addition of a second adjacent helix has no influence on the lipid affinity of the apoA-I 145-183 peptide compared to the shorter segments but substantially improves the stability of the complexes. The helical content of the peptide increases upon lipid association as observed with apoA-I. The complexes generated with the apoA-I 145-183 peptide appear as discoidal particles by negative staining electron microscopy, with heterogeneous sizes ranging between 250 and 450 A. The relative orientation of the peptide and the phospholipid is the same as in a DMPC/apoA-I complex as the helices are oriented parallel to the acyl chains of the phospholipid. However, the stability of these complexes is significantly lower than that of the corresponding DMPC/apoA-I complexes. The transition temperature, fluidity, and cooperativity of the phospholipid bilayer are only weakly affected by the association with the apoA-I 145-183 peptide. These data suggest that a pair of helical peptides linked through a beta-strand associates more tightly with lipids and can form discoidal lipid-peptide complexes, than a single helix. A comparison with the properties of native apoA-I suggests, however, that the cooperativity between pairs of helices in native apoA-I further contributes to strengthen the lipid-protein association.