Examining Properties of the C-Terminal Domain of Human Apolipoprotein A-I Utilizing A Chimeric Apolipoprotein

Human apolipoprotein A-I (apoA-I) is a 28 kDa exchangeable apolipoprotein that constitutes the primary protein of high density lipoprotein and plays a vital role in reverse cholesterol transport. ApoA-I comprises two structural domains: an α-helical N-terminal domain (NT, residues 1-189) and a less structured C-terminal domain (CT, residues 190-243). To evaluate the properties of the CT domain of apoA-I independently from the NT domain, a chimeric apolipoprotein was engineered in which residues 179-243 of apoA-I were attached to apolipophorin III (apoLp-III). ApoLp-III is a well-characterized insect apolipoprotein with a one domain structure composed of a bundle of α-helices. Two cysteine residues were introduced in apoLp-III to control opening of the helix bundle. ApoLp-III is monomeric but cross-linking studies with dimethyl suberimidate showed that the chimera self-associates similar to apoA-I. Far UV circular dichroism showed that the chimera displayed less α-helical content compared to apoLp-III, which is consistent with the addition of a less structured domain. Denaturation analysis using guanidine HCl showed that the stability of the chimera increased compared to apoLp-III. Functional analysis showed that the chimera was able to solubilize phospholipid vesicles at a faster rate than apoLp-III and that the percent calcein released from large unilamellar vesicles made of phosphatidylglycerol was substantially higher. Oxidized chimera protected LDL from phospholipase C-induced aggregation to a similar extent as apoLp-III but was far superior in its reduced state. Since the attachment of the CT domain of apoA-I to apoLp-III resulted in oligomerization and enhanced lipid binding, this demonstrated that these properties reside in the CT domain of apoA-I, and were transferable to an unrelated apolipoprotein. This demonstrates the potential to use the chimera as a tool to study the CT domain of apoA-I.