Analysis of Unfolding of Apolipoprotein E Offers Insights into Lipid Binding Mechanism

Apolipoprotein E (apoE) is a cholesterol transport protein that plays a critical role in lipid homeostasis. It is composed of a series of amphipathic α-helices: H1-H4 in the N-terminal (NT), and C1-C3 in the C-terminal (CT) domain. In humans, those bearing the APOE e4 allele are at a higher risk for developing heart disease and Alzheimer's disease than those with APOE e3 allele. The goal is to understand isoform specific differences in behavior between apoE3 and apoE4. The objectives are to compare their unfolding behavior at the sub-domain level, and, to determine the order of helix unfolding. Recombinant apoE bearing single Cys at defined locations were generated for labeling with fluorophores. Chemical-induced unfolding was monitored by circular dichroism and fluorescence spectroscopy. Fluorescence polarization measurements were carried out to determine changes in probe mobility. With apoE3 NT domain, helices H3 and H4 unfold with a significantly lower mid point of denaturation compared to H1 and H2, whereas with the CT domain, helix C3 offers the least resistance to unfolding compared to C1 and C2. Studies with the intact protein suggest a bi-phasic unfolding for apoE3, where the two domains unfold independently, and a cooperative unfolding for apoE4 possibly due to interaction between helices in the NT and CT domains. Taken together, our studies suggest the order of unfolding of helices: C3-C1/C2-H3/H4-H1/H2. We propose that apoE3 follows a similar order of lipid binding, based on the rationale that the ease of unfolding is reflective of the ease of lipid binding, with less structured segments seeking stability by lipid interaction. Our data are significant because understanding isoform-specific differences at the molecular level allows us to obtain insights into the physiological behavior of apoE in lipid metabolism.