Examination of lipid-bound conformation of apolipoprotein E4 by pyrene excimer fluorescence.
Apolipoprotein E (apoE) is a 34-kDa resident of lipoproteins that plays a key role in cholesterol homeostasis in plasma and in brain. It is composed of an N-terminal (NT) domain (residues 1-191) and a C-terminal (CT) domain (residues 201-299). Of the three major isoforms (apoE2, -E3, and -E4), apoE4 is considered a risk factor for both cardiovascular and Alzheimer disease. Compared with apoE3, domain interaction between NT and CT domains is believed to direct the lipoprotein distribution preference of apoE4 for very low density lipoprotein-sized particles. We examined the relative disposition of apoE4 NT and CT domains in lipid-free and lipid-bound forms by monitoring pyrene excimer fluorescence emission as a direct indicator of spatial proximity. Site-specific labeling of apoE4 by N-(1-pyrene)maleimide was accomplished after substitution of Cys residues for Arg-61 in NT domain and Glu-255 in CT domain. Pyrene labeling did not alter the lipoprotein distribution pattern of apoE4 in plasma. Pyrene excimer fluorescence was noted in lipid-free pyrene-R61C/E255C/apoE4 in mixtures containing excess wild-type apoE4, which was attributed to intramolecular spatial proximity between these specified sites. Upon disruption of tertiary interaction, a large decrease in excimer fluorescence emission was noted in pyrene-R61C/E255C/apoE4. In dimyristoylphosphatidylcholine/pyrene-R61C/E255C/apoE4 discoidal complexes, pyrene excimer fluorescence emission was retained. Taken together with fluorescence quenching and cross-linking analysis, a looped-back model of apoE4 is proposed in lipid-bound state, including spherical lipoprotein particles, wherein residues Arg-61 and Glu-255 are proximal to one another.
Drury
Jessica
J
Lipid Biology in Health and Disease Research Group, Children's Hospital Oakland Research Institute, Oakland, California 94609, USA.
Narayanaswami
Vasanthy
V
D053327:Apolipoprotein E4; D001057:Apolipoproteins E / Q000737:chemistry*; D001120:Arginine / Q000737:chemistry; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D031541:Fluorescence Resonance Energy Transfer; D018698:Glutamic Acid / Q000737:chemistry; D006801:Humans; D008055:Lipids / Q000737:chemistry*; D008074:Lipoproteins / Q000737:chemistry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D011721:Pyrenes / Q000737:chemistry*; D012307:Risk Factors; D013050:Spectrometry, Fluorescence
D053327:Apolipoprotein E4; D001057:Apolipoproteins E; D003432:Cross-Linking Reagents; D008055:Lipids; D008074:Lipoproteins; D020033:Protein Isoforms; D011721:Pyrenes; D018698:Glutamic Acid; D001120:Arginine; D003545:Cysteine