A Monomeric, Biologically Active, Full-Length Human Apolipoprotein E† Export

@article{citeulike:6083691, abstract = {PMID: 17715945 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 95100\% 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 domaindomain 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 192215) that connects apoE's N- and C-terminal domains may play an important role in mediating this domaindomain interaction.}, author = {Zhang, Yonghong and Vasudevan, Sheeja and Sojitrawala, Radiya and Zhao, Wentao and Cui, Chunxian and Xu, Chao and Fan, Daping and Newhouse, Yvonne and Balestra, Reeny and Jerome, W. Gray and Weisgraber, Karl and Li, Qianqian and Wang, Jianjun}, citeulike-article-id = {6083691}, citeulike-linkout-0 = {http://dx.doi.org/10.1021/bi700672v}, citeulike-linkout-1 = {http://pubs.acs.org/doi/abs/10.1021/bi700672v}, day = {1}, doi = {10.1021/bi700672v}, journal = {Biochemistry}, keywords = {a, apolipoprotein, e, monomeric, yonghong, zhang}, month = {September}, number = {37}, pages = {10722--10732}, posted-at = {2009-11-08 06:54:38}, priority = {2}, title = {A Monomeric, Biologically Active, Full-Length Human Apolipoprotein E{\dag}}, url = {http://dx.doi.org/10.1021/bi700672v}, volume = {46}, year = {2007} }

TY - JOUR ID - citeulike:6083691 L3 - citeulike-article-id:6083691 N2 - PMID: 17715945 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 95100% 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 domaindomain 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 192215) that connects apoE's N- and C-terminal domains may play an important role in mediating this domaindomain interaction. IS - 37 JF - Biochemistry EP - 10732 TI - A Monomeric, Biologically Active, Full-Length Human Apolipoprotein E† VL - 46 SP - 10722 KW - a KW - apolipoprotein KW - e KW - monomeric KW - yonghong KW - zhang AU - Zhang, Yonghong AU - Vasudevan, Sheeja AU - Sojitrawala, Radiya AU - Zhao, Wentao AU - Cui, Chunxian AU - Xu, Chao AU - Fan, Daping AU - Newhouse, Yvonne AU - Balestra, Reeny AU - Jerome, Gray AU - Weisgraber, Karl AU - Li, Qianqian AU - Wang, Jianjun PY - 2007/09/01/ UR - http://dx.doi.org/10.1021/bi700672v ER -