Conformational studies of human apolipoprotein E3 as revealed by fluorescence polarization and mass spectrometry

Apolipoprotein E3 (apoE3) is an important anti-atherogenic protein that helps maintain cholesterol levels in the brain and plasma. It is responsible for binding and cellular uptake of plasma lipoproteins via the low-density lipoprotein receptor family of proteins. Fluorescence intensity measurements demonstrate environmentally sensitive fluorescent probes undergoing bathochromic shift due to an increase in polar environment. Polarization studies indicate that the unfolding is likely initiated at the C-terminal end of the protein, the CT domain unfolds prior to NT domain, and that the NT domain forms a highly stable helix bundle. Hydrogen deuterium exchange mass spectrometry analysis revealed that the amide backbone of the NT domain underwent helix specific exchange where helix 1 and 2 have higher percent deuterium compared to helix 3 and 4. In contrast, the CT domain revealed significantly higher HDX rates. Our studies suggest that the two domains of apoE may undergo independent conformational reorganization.