Structural studies of a consensus sequence peptide (CSP) ABAB of apolipoproteins through NMR spectroscopy
The apolipoproteins play critical roles in lipid transport, lipid metabolism and the pathophysiology of dyslipoproteinemias, most importantly atherosclerosis. ApoA-1 is a representative member of the family of exchangeable apolipoproteins and the major apolipoprotein of high density lipoprotein (HDL). HDL is responsible for the pathway of reverse cholesterol transport and the only particle capable of removing cholesterol from peripheral cells for transport to the liver. The sequences ofthe exchangeable apolipoproteins contain 11/22 residue tandem sequence repeats forming amphipathic α-helices that are believed to be responsible for lipid binding. The consensus sequence peptide (CSP) for this repeat was derived based on the characteristic residue distribution of the exchangeable apolipoproteins. The derived consensus sequence containing motifs A, (PLAEELRARLR), and B, (AQLEELRERLG), represent an idealized lipid binding model and fundamental structural motif of the exchangeable apolipoproteins. The recombinant CSP-ABAB peptide was successfully expressed in E. coli and purified. Circular dichroism showed that CSP-ABAB is ~62% α-helical, i.e.~27 residues of 44 residues are in helical conformation. The CSP-ABAB peptide was successfully 15N, 13C labeled and the detailed tertiary structure was explored by NMR spectroscopy. The peptide's backbone and side-chain resonances were successfully assigned and ten water refined structural conformers of CSP-ABAB were generated. The ten structural conformers all employ anti-parallel helical conformation in solution. Hydrophobic inter-helical interactions play a major role to stabilize the antiparallel helical hairpin conformation. There are also intra-/inter-helical salt bridges present on the surface of the CSP-ABAB molecule providing additional stabilization. The structural features of the NMR structures suggest a lipid binding model of CSP-ABAB. When lipids are introduced, the exposed hydrophobic ridge contributed by the twelve leucine residues firstly bind to the lipids. At the same time, a hydrophobic concave surface created by the four alanine residues at the center of the interface is accessed by the introduced lipids. These two steps open the anti-parallel helical hairpin conformation to form a fully extended α-helix. Similar hydrophobic inter-helical stabilization interactions and new intra-/inter-helical salt bridges between two different CSP-ABAB molecules are reformed to stabilize the 'double-belt' arrangement. This lipid binding model of CSP-ABAB sheds light on the lipid binding of apoA-I and the mechanism of HDL formation.
Thesis (Ph.D.)--Boston University