Fluorescence studies of exchangeable apolipoprotein-lipid interactions - Superficial association of apolipophorin III with lipoprotein surfaces

Apolipophorin III (apoLp-III) from the Sphinx moth, Manduca sexta, is an 18-kDa exchangeable apolipoprotein that reversibly associates with lipoprotein particles, In the absence of Lipid, apoLp-III exists as an elongated bundle of five amphipathic alpha-helices. Upon lipid association, the protein is postulated to undergo a major conformational change, wherein the bundle opens around hinge loop regions, resulting in exposure of its hydrophobic interior, Fluorescence quenching techniques have been employed to study apoLp-III helix topography and spatial arrangement in phospholipid disc complexes and intact lipoprotein particles, Intrinsic fluorescence of the single tyrosine in apoLp-III was exploited to monitor the location of helix 5 in model disc complexes, To investigate other regions of the protein, site-directed mutagenesis was performed to introduce cysteine residues, replacing Asn-40 (helix 2, N40C) or Leu-90 (helix 3, L90C), thereby providing two mutant apoLp-IIIs, each with a single site for covalent attachment of the extrinsic fluorescent probe, N-(l-pyrene) maleimide, In the lipid-free state, pyrene-N40C- and pyrene-L90C-apoLp-III were highly accessible to the negatively charged aqueous quencher KI, yielding K-sv, values of 27.1 and 19.8 M-1, respectively, Upon binding to the surface of a spherical lipoprotein particle, K-sv, values for hi decreased by about 90% for bath pyrene-labeled apoLp-IIIs, indicating a significant change in the local microenvironment of the fluorophores. A lesser decrease in K-sv,, was observed when the pyrene-labeled apoLp-IIIs were bound to phospholipid disc complexes. When spin-labeled fatty acids 5-doxylstearic acid and 12-doxylstearic acid were used as lipophilic quenchers, tyrosine and pyrene fluorescence were more effectively quenched by 5-doxylstearic acid in both phospholipid bilayer disc complexes and spherical lipoprotein particles., These data provide insight into the spatial topography of apoLp-III alpha-helices in phospholipid disc complexes and support the concept that interaction with spherical lipoprotein particles results in superficial contact of apoLp-III helical segments with the monolayer surface, providing a basis for its reversible binding ability.