ASSOCIATION OF SYNTHETIC PEPTIDE-FRAGMENTS OF HUMAN APOLIPOPROTEIN-A-I WITH PHOSPHOLIPIDS

The sequences of the plasma apolipoproteins have a high degree of inte rnal homology as they contain several 22-mer internal repeats. These a mphipathic helical repeats are considered as the structural and functi onal units of this class of proteins. We proposed that the 22-mer repe ats of the plasma apolipoproteins consist of 17-mer helical segments s eparated by extended beta-strands comprising five amino acid residues with a proline in the center of this segment. These beta-strand segmen ts help reverse the orientation of the consecutive helices of apoA-I, A-IV, and E in a discoidal apolipoprotein-phospholipid complex. In ord er to support this hypothesis, we synthesized apoA-I fragments consist ing of, respectively, one putative helix (residues 166-183), one helix plus a beta-strand (residues 161-183), and a pair of helices separate d by a beta-strand (residues 145-183). The structural and lipid-bindin g properties of these peptides were investigated by turbidity, fluores cence, binding studies with unilamellar phospholipid vesicles, electro n microscopy, and circular dichroism measurements. Our data show that one single putative helical segment or one helical segment plus one ex tended beta-strand do not form stable complexes with phospholipids. Th e addition of a second adjacent helix has no influence on the lipid af finity 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 w ith apoA-I. The complexes generated with the apoA-I 145-183 peptide ap pear as discoidal particles by negative staining electron microscopy, with heterogeneous sizes ranging between 250 and 450 A. The relative o rientation of the peptide and the phospholipid is the same as in a DMP C/apoA-I complex as the helices are oriented parallel to the acyl chai ns of the phospholipid. However, the stability of these complexes is s ignificantly lower than that of the corresponding DMPC/apoA-I complexe s. The transition temperature, fluidity, and cooperativity of the phos pholipid bilayer are only weakly affected by the association with the apoA-I 145-183 peptide. These data suggest that a pair of helical pept ides 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, th at the cooperativity between pairs of helices in native apoA-I further contributes to strengthen the lipid-protein association.