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 associ ation, the protein is postulated to undergo a major conformational cha nge, wherein the bundle opens around hinge loop regions, resulting in exposure of its hydrophobic interior, Fluorescence quenching technique s have been employed to study apoLp-III helix topography and spatial a rrangement in phospholipid disc complexes and intact lipoprotein parti cles, Intrinsic fluorescence of the single tyrosine in apoLp-III was e xploited to monitor the location of helix 5 in model disc complexes, T o 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 extrinsi c fluorescent probe, N-(l-pyrene) maleimide, In the lipid-free state, pyrene-N40C- and pyrene-L90C-apoLp-III were highly accessible to the n egatively charged aqueous quencher KI, yielding K-sv, values of 27.1 a nd 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 b ath 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 phosph olipid disc complexes. When spin-labeled fatty acids 5-doxylstearic ac id and 12-doxylstearic acid were used as lipophilic quenchers, tyrosin e and pyrene fluorescence were more effectively quenched by 5-doxylste aric acid in both phospholipid bilayer disc complexes and spherical li poprotein particles., These data provide insight into the spatial topo graphy of apoLp-III alpha-helices in phospholipid disc complexes and s upport the concept that interaction with spherical lipoprotein particl es results in superficial contact of apoLp-III helical segments with t he monolayer surface, providing a basis for its reversible binding abi lity.