| Exchangeable apolipoproteins form a family of proteins that play critical roles in lipoprotein metabolism and many other biological processes. The lack of high-resolution structural information hinders the structure-functional studies of this important protein family. Nuclear Magnetic Resonance (NMR) spectroscopy has been demonstrated to be a powerful tool for the structural determination of apolipoproteins.;Apolipoprotein E is a 299-residue, two-domain protein. The C-terminal domain is the major lipoprotein-binding domain and is also responsible for the aggregation of apoE, which is the major challenge for the structural determination of apoE and the C-terminal domain. Using protein engineering techniques, we detected that a cluster of hydrophobic residues in the region of residues 257–287 mediate apoE's aggregation in an additive manner. By mutating five of those residues to Ala or charged/ionizable residues, we generated a monomeric and biologically active apoE C-terminal domain mutant. Using NMR spectroscopy, we determined the high-resolution structure of this apoE C-terminal domain mutant in a DPC-micelle-bound form. The apoE C-terminal domain adopts a curved long α-helix with the hydrophobic concave face orientated towards the DPC-micelle.;Locusta migratoria apolipophorin-III is a 164-residue insect apolipoprotein which serves a prototype for structural-functional studies of exchangeable apolipoproteins. Although its x-ray structure was determined in 1991, there are a few structure-related issues remaining to be clarified. Using NMR spectroscopy, we determined the solution structure of L -apoLp-III in the lipid-free state and measured its backbone dynamics. Based on the solution structure and dynamics information, we proposed a molecular model for the apoLp-III helix-bundle opening and recovery up lipid-binding and dissociation.;LDL receptor-associated protein (RAP) is a 323-residue ER-resident chaperone molecule for the LDL receptor family. Using protein cross-linking, circular dichroism and NMR spectroscopy, we characterized the structural features of this protein and its fragments. Our results indicated that RAP consists of three independently-folded domains, each of them roughly corresponds to one of the three sequence repeats; the third domain adopts a flexible conformation without receptor, but becomes a well-defined bound conformation in the presence of receptor. We also demonstrated that RAP is a phospholipid binding protein and this activity may be relevant to its functionality. |
