title: A monomeric, biologically active, full-length human apolipoprotein E.
abstract: Apolipoprotein E (apoE) is an exchangeable apolipoprotein that plays an important role in lipid/lipoprotein metabolism and cardiovascular diseases. Recent evidence indicates that apoE is also critical in several other important biological processes, including Alzheimer's disease, cognitive function, immunoregulation, cell signaling, and infectious diseases. Although the X-ray crystal structure of the apoE N-terminal domain was solved in 1991, the structural study of full-length apoE is hindered by apoE's oligomerization property. Using protein-engineering techniques, we generated a monomeric, biologically active, full-length apoE. Cross-linking experiments indicate that this mutant is nearly 95-100% monomeric even at 20 mg/mL. CD spectroscopy and guanidine hydrochloride denaturation demonstrate that the structure and stability of the monomeric mutant are identical to wild-type apoE. Monomeric and wild-type apoE display similar lipid-binding activities in dimyristoylphosphatidylcholine clearance assays and formation of reconstituted high-density lipoproteins. Furthermore, the monomeric and wild-type apoE proteins display an identical LDL receptor binding activity. Availability of this monomeric, biologically active, full-length apoE allows us to collect high quality NMR data for structural determination. Our initial NMR data of lipid-free apoE demonstrates that the N-terminal domain in the full-length apoE adopts a nearly identical structure as the isolated N-terminal domain, whereas the C-terminal domain appears to become more structured than the isolated C-terminal domain fragment, suggesting a weak domain-domain interaction. This interaction is confirmed by NMR examination of a segmental labeled apoE, in which the N-terminal domain is deuterated and the C-terminal domain is double-labeled. NMR titration experiments further suggest that the hinge region (residues 192-215) that connects apoE's N- and C-terminal domains may play an important role in mediating this domain-domain interaction.
authors: Department of Biochemistry and Molecular Biology, School of Medicine, Wayne State University, Detroit, Michigan 48201, USA.
authors: Yonghong
authors: Y
authors: Zhang
authors: Sheeja
authors: S
authors: Vasudevan
authors: Radiya
authors: R
authors: Sojitrawala
authors: Wentao
authors: W
authors: Zhao
authors: Chunxian
authors: C
authors: Cui
authors: Chao
authors: C
authors: Xu
authors: Daping
authors: D
authors: Fan
authors: Yvonne
authors: Y
authors: Newhouse
authors: Reeny
authors: R
authors: Balestra
authors: W Gray
authors: WG
authors: Jerome
authors: Karl
authors: K
authors: Weisgraber
authors: Qianqian
authors: Q
authors: Li
authors: Jianjun
authors: J
authors: Wang
mesh_terms: D053318:Apolipoprotein E3 / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D002942:Circular Dichroism; D004134:Dimyristoylphosphatidylcholine / Q000378:metabolism; D006801:Humans; D050356:Lipid Metabolism; D008075:Lipoproteins, HDL / Q000648:ultrastructure; D009682:Magnetic Resonance Spectroscopy; D050505:Mutant Proteins / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D011973:Receptors, LDL / Q000378:metabolism; D013329:Structure-Activity Relationship; D013816:Thermodynamics
chemical_list: D053318:Apolipoprotein E3; D008075:Lipoproteins, HDL; D050505:Mutant Proteins; D011973:Receptors, LDL; D004134:Dimyristoylphosphatidylcholine