Interaction of the α-Helices of Apolipophorin III with the Phospholipid Acyl Chains in Discoidal Lipoprotein Particles:  A Fluorescence Quenching Study^†

Abstract

Quenching of tryptophan fluorescence by nitroxide-labeled phospholipids and nitroxide-labeled fatty acids was used to investigate the lipid-binding domains of apolipophorin III. The location of the Trp residues relative to the lipid bilayer was investigated in discoidal lipoprotein particles made with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and five different single-Trp mutants of apoLp-III. A comparison of the quenching efficiencies of phospholipids containing nitroxide groups at the polar head, and at positions 5 and 16 of the sn-2 acyl chain, indicated that the protein is interacting with the acyl chains of the phospholipid along the periphery of the bilayer of the discoidal lipoprotein. N-Bromosuccinimide readily abolished 100% of the fluorescence of all Trp residues in the lipid-bound state. Larger quenching rates were observed for the Trp residues in helices 1, 4, and 5 than for those located in helices 2 and 3, suggesting differences between the interaction of these two groups of helices. However, the extent of Trp fluorescence quenching observed in lipoproteins made with any of the mutants was comparable to that reported for deeply embedded Trp residues, suggesting that all Trp residues interact with the phospholipid acyl chains. This study provides the first experimental evidence of a massive interaction of the alpha-helices of apoLp-III with the phospholipid acyl chains in discoidal lipoproteins. The extent of interaction deduced is consistent with the apolipoprotein adopting a highly extended conformation.

Structure of Apolipophorin-III in Discoidal Lipoproteins

“…The extended conformation is directly supported by the long distances separating the centers of the pairs of helices 3-4 and 4 -5 as well as by the fact that the estimates for the separation distance between the helix pairs 1-4 and 2-4 are at the limit of the maximum distances that can be estimated by FRET, suggesting than even longer distances may be possible. The extended conformation is also compatible with recent studies indicating that in discoidal lipoproteins all helices of apoLp-III appear to interact with the phospholipid acyl chains (18). Moreover, the extended conformation is consistent with a previous report showing that a disulfide mutant tethering the helices 1 and 5 (22) is unable to interact with liposomes of phosphatidylcholine, leading to the spontaneous formation of discoidal lipoproteins.…”

“…Additional experimental evidence about the topology of apoLp-III in discoidal lipoproteins was recently presented in a fluorescence-quenching study. The quenching pattern of single-Trp mutants obtained with phospholipids containing quenchers at different depths was shown to be consistent with a location of apoLp-III around the periphery of the disc bilayer (18).…”

“…Thus, a fully extended polypeptide chain would produce essentially the same results that were observed in this study. In fact, on the basis of the results of this study as well as on the conclusions of recent studies reporting on the topology and lipid-interacting regions of the apolipoprotein (17,18), a structural model of apoLp-III in discoidal lipoproteins is proposed below. In this model it is assumed that the apolipoprotein adopts a fully extended helical conformation.…”

“…The first of these studies, which used water-soluble quenchers, suggested that at least three of the five ␣-helices (helices 1, 4, and 5) interact with the lipid surface (17). The second study, which used lipid quenchers, suggested that in discoidal lipoproteins all ␣-helices of apoLp-III interact with the phospholipid acyl chains (18). Distances H1-H4, H2-H4, H3-H4, and H5-H4 -The distance between the centers of helices 5 and 4 ( 40 Å) estimated in this study is consistent with a collinear arrangement of these two helices in the lipid-bound state.…”

Purification and Characterization of Recombinant Lipid Storage Protein-2 from Drosophila melanogaster

“…Lipoprotein complexes of Lsd 2 and DMPG were obtained by the cholate dialysis method amply used to obtain lipoprotein particles with apolipoproteins [18] and recently used to obtain phospholipid-Lsd1complexes [13]. Lsd2 formed small lipoprotein complexes suitable for spectroscopic studies.…”