Faculty Collaboration Database

Fluorescence studies of exchangeable apolipoprotein-lipid interactions. Superficial association of apolipophorin III with lipoprotein surfaces. J Biol Chem 1998 Jan 16;273(3):1403-8

Date

01/27/1998

Pubmed ID

9430675

DOI

10.1074/jbc.273.3.1403

Scopus ID

2-s2.0-0031914422 (requires institutional sign-in at Scopus site)   20 Citations

Abstract

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.

Author List

Sahoo D, Narayanaswami V, Kay CM, Ryan RO

Author

Daisy Sahoo PhD Dean, Professor in the Medicine department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Alkylation
Animals
Apolipoproteins
Carrier Proteins
Lipoproteins
Lipoproteins, LDL
Manduca
Mutagenesis, Site-Directed
Nephelometry and Turbidimetry
Protein Conformation
Protein Structure, Secondary
Spectrometry, Fluorescence
Structure-Activity Relationship
Surface Properties
Type C Phospholipases