Structural Analysis of Human Apolipoprotein E3 by Fluorescence Spectroscopy, and Hydrogen/Deuterium Exchange Coupled to 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. It is a highly alpha-helical protein that can exist in lipid-free and lipid-bound states. ApoE3 is composed of two domains in lipid-free state: an N-terminal (NT) domain folded into a 4-helix bundle and a C-terminal (CT) domain that mediates apoE3 oligomerization via inter-molecular helix-helix interactions. The objective of this study is to understand the conformational organization of lipid-free apoE in its oligomeric state. We employed chemical-induced denaturation coupled to fluorescence spectroscopy of apoE bearing environmentally sensitive fluorescent probes monitoring different helices in the two domains, to obtain information regarding tertiary conformation. in a complementary approach, we also used hydrogen/deuterium exchange coupled to mass spectrometry (HDX-MS) to understand amide-backbone structural dynamics, solvent accessibility, and helical contours of apoE3. Fluorescence intensity and 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. HDX/MS analysis revealed that the amide backbone of the NT domain underwent limited exchange, with the exception of the first 14 residues at the N-terminal end, and, those linking helices 2 and 3 (79-93), confirming the presence of a tight helix bundle. In contrast, the CT domain revealed significantly higher HDX rates. Our studies suggest that the two domains of apoE may undergo independent conformational reorganization, a concept that bears significant relevance in terms of apoE interaction with lipids and lipoproteins. NIH-HL096365 and TRDRP 17RT-0165.