Conformation of apolipoprotein E both in free and in lipid-bound form may determine the avidity of triglyceride-rich lipoproteins to the LDL receptor: structural and kinetic study

Abstract

Slow refolding of human apolipoprotein E (apoE) in solution after guanidine- or cholate-induced denaturation followed by dialysis under controlled conditions was investigated using various spectroscopic properties of fluorescein- and dansyl-labeled apolipoprotein molecules. The results suggest that the last phase(s) of apoE refolding in solution include a slow (several hours at 24 degrees C) interconversion of a self-associated 'open' conformer into a more dense 'closed' conformer. The hydrophobic interactions are primarily responsible for the formation of this more compact apoE structure. To visualize the contribution of apolipoprotein conformation and/or the number of 'active' lipid-bound apoE molecules in the reaction of binding to the low density lipoprotein receptor (LDLr) by solid-phase binding assay, the complexes of human plasma apolipoprotein or recombinant (rec) apoE3 with dipalmitoylphosphatidylcholine (DPPC) or palmitoyloleoylphosphatidylcholine (POPC) varying in size were used. For seven complexes with plasma protein (four DPPC and three POPC complexes), the final phosphatidylcholine (PC)/protein mole ratio ranged from 117 to 279; affinity constant K(a) averaged for both PCs and plotted against this ratio abruptly increased from 3.8 x 10(7) to 3.8 x 10(8) M(-1) with a transition midpoint of 150-180 PC/apoE, mole ratio. Two DPPC complexes with rec protein bind much more efficiently. Complexes with both plasma and rec apoE were able to compete with very low density…