title: A monomeric human apolipoprotein E carboxyl-terminal domain.
abstract: ApoE plays a critical role in lipoprotein metabolism and plasma lipid homeostasis through its high-affinity binding to the LDL-receptor family. In solution, apoE is an oligomeric protein and the C-terminal domain causes apoE's aggregation. The aggregation property presents a major difficulty for the structural determination of this protein. A high-level expression system of the apoE C-terminal domain is reported here. Using protein engineering techniques, we identified a monomeric, biologically active apoE C-terminal domain mutant. This mutant replaces five bulky hydrophobic residues in the region of residues 253-289 with either smaller hydrophobic or polar/charged residues (F257A, W264R, V269A, L279Q, and V287E). The solubility of the mutant is significantly increased ( approximately 10-fold). Cross-linking experiments indicate that this mutant is 100% monomeric even at 5 mg/mL. CD and guanidine hydrochloride denaturation results indicate that the mutant maintains an identical alpha-helical secondary structure and stability as compared with those of the wild-type protein. DMPC-binding assays demonstrate an identical vesicle clearance rate shared by both the mutant and the wild-type apoE C-terminal domain. In addition, electron microscopic results show identical recombinant HDL particles prepared with both the mutant and the wild-type proteins. These results indicate that residues F257, W264, V269, L279, and V287 are critical residues for aggregation but may not be important in maintaining the structure, stability, and lipid-binding activity of this apoE domain, suggesting that apoE may use different "epitopes" for its aggregation property, helical structure/stability, and lipid-binding activity. Finally, preliminary NMR data demonstrated that we have collected high-quality NMR spectra, allowing for an NMR structural determination of the apoE C-terminal domain.
authors.affiliation: Department of Biochemistry and Molecular Biology, Southern Illinois University at Carbondale, Carbondale, Illinois 62901-4413, USA.
authors.forename: Daping
authors.initials: D
authors.lastname: Fan
authors.forename: Qianqian
authors.initials: Q
authors.lastname: Li
authors.forename: Leslie
authors.initials: L
authors.lastname: Korando
authors.forename: W Gray
authors.initials: WG
authors.lastname: Jerome
authors.forename: Jianjun
authors.initials: J
authors.lastname: Wang
mesh_terms: D000595:Amino Acid Sequence; D001057:Apolipoproteins E / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics; D002942:Circular Dichroism; D017124:Conserved Sequence; D003432:Cross-Linking Reagents / Q000737:chemistry; D004134:Dimyristoylphosphatidylcholine / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D019791:Guanidine / Q000494:pharmacology; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008075:Lipoproteins, HDL / Q000648:ultrastructure; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D011489:Protein Denaturation; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000648:ultrastructure; D017386:Sequence Homology, Amino Acid; D012995:Solubility
chemical_list: D001057:Apolipoproteins E; D003432:Cross-Linking Reagents; D008075:Lipoproteins, HDL; D011994:Recombinant Proteins; D019791:Guanidine; D004134:Dimyristoylphosphatidylcholine