A monomeric human apolipoprotein E carboxyl-terminal domain.

Fan D, Li Q, Korando L, Jerome WG, Wang J
Biochemistry. 2004 43 (17): 5055-64

PMID: 15109264 · DOI:10.1021/bi035958w

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.

MeSH Terms (22)

Amino Acid Sequence Apolipoproteins E Circular Dichroism Conserved Sequence Cross-Linking Reagents Dimyristoylphosphatidylcholine Escherichia coli Guanidine Humans Hydrophobic and Hydrophilic Interactions Lipoproteins, HDL Models, Molecular Molecular Sequence Data Mutation Nuclear Magnetic Resonance, Biomolecular Protein Denaturation Protein Engineering Protein Structure, Secondary Protein Structure, Tertiary Recombinant Proteins Sequence Homology, Amino Acid Solubility

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