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.