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-(1-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 Ksv values of 27.1 and 19.8 M-1, respectively. Upon binding to the surface of a spherical lipoprotein particle, Ksv values for KI decreased by about 90% for both pyrene-labeled apoLp-IIIs, indicating a significant change in the local microenvironment of the fluorophores. A lesser decrease in Ksv 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 apoL-III helical segments with the monolayer surface, providing a basis for its reversible binding ability.
Sahoo
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Department of Biochemistry, University of Alberta, Edmonton, Canada.
Narayanaswami
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Kay
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Ryan
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D000478:Alkylation; D000818:Animals; D001053:Apolipoproteins / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D008074:Lipoproteins / Q000378:metabolism*; D008077:Lipoproteins, LDL / Q000378:metabolism; D018402:Manduca; D016297:Mutagenesis, Site-Directed; D009391:Nephelometry and Turbidimetry; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013499:Surface Properties; D010738:Type C Phospholipases / Q000378:metabolism
D001053:Apolipoproteins; D002352:Carrier Proteins; D008074:Lipoproteins; D008077:Lipoproteins, LDL; C042470:apolipophorin III; D010738:Type C Phospholipases