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. ApoE is a polymorphic protein with three major isoforms, apoE2, apoE3 and apoE4. The apoE isoforms differ from one another only by a single amino acid substitution, yet they have profound functional consequences at both the cellular and molecular levels. Although the X-ray crystal structure of the apoE N-terminal domain was solved in 1991, the structural studies of full-length apoE and the apoE C-terminal domain is hindered by apoE's oligomerization property. It is well established that the C-terminal domain causes apoE aggregation. A monomeric, biologically active apoE C-terminal domain has been generated in our laboratory recently, which has solved the major technical problem in the apoE structural study. The NMR spectra of this monomeric, biologically active apoE C-terminal domain has been completely assigned and its NMR structure will be solved soon. This progress places us in a very good position to propose a NMR structural determination of full-length apoE. This research proposal focuses on solving the NMR structure of full-length human apoE in the lipid-free state using nuclear magnetic resonance (NMR) and molecular biology techniques. In addition, a high-level expression and refolding system has been established for the LDL receptor ligand-binding domain repeats (LDLR-LBDR) and LRP ligand binding domain 2 repeats (LRP-LBD2R), allowing us to propose to characterize the structural changes of the binding residues in apoE upon interaction with receptors. Finally, identification of the critical residues that are involved in the apoE domain-domain interactions has also been proposed in this proposal. Due to the importance of apoE and the apoE/receptor interactions in several major human diseases, including atherosclerosis and Alzheimer's disease, the significance of this proposal is very well justified. It is worth noting that one unique feature of this application is that several independent approaches have been proposed for each specific objective, ensuring that any success in one approach will achieve the overall goal of this objective. ? ?
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