PROJECT SUMMARY A single amino acid difference in apolipoprotein E (apoE) distinguishes each of the three major isoforms that lead to dramatically different functional outcomes when expressed in the brain: apoE4 contributes up to a 15 fold risk increase in Alzheimer’s disease (AD) compared to apoE3, whereas apoE2 appears to be protective. A large amount of evidence suggests a direct role of the protein in disease progression; yet, the isoform- dependent structural features of apoE remain elusive, hampering our understanding of the mechanism behind apoE4 neurotoxicity. Indeed, current structural models of apoE have been derived from a limited number of incomplete structures obtained from protein fragments free in solution or in complex with synthetic liposomes. Very little is known about the structure in the context of endogenously-secreted lipoproteins. Testing the ApoE Cascade Hypothesis (ACH), the core focus of this U19 proposal, requires overcoming these limitations and comparing the structural differences of apoE isoforms when bound to secreted lipoproteins and AD-related pathogenic molecules such as amyloid-β (Aβ). Here, we propose to bypass previous experimental obstacles by using an innovative multipronged approach that combines single- molecule fluorescence spectroscopy and cryo- electron microscopy (EM). Our approach enables accessing the conformational ensemble of apoE in its monomeric, oligomeric, and lipid-bound forms and reveals coexistence of multiple conformations in equilibrium, which are invisible to classical methods of structural biology. In collaboration with Core B, single-molecule measurements, negative-stain, and cryo-EM will be compared and interpolated with molecular dynamics simulations to reconstruct atomistic-detailed models of the protein when bound to secreted lipoproteins and Aβ. In Aim 1, we will determine the isoform-specific structural ensemble of apoE/lipoprotein particles obtained from human apoE knockin mouse-derived astrocytes, microglia, and vascular mural cells (Project 2 and 4), as well as iPSC-derived cells (Core E), cell type-specific apoE mouse models (Projects 3-4), and human biospecimens (Core C and D). In aim 2, we will assess how the interplay between Aβ and apoE modulate their structural ensembles and oligomerization propensity. The proposed experiments will provide a detailed atomistic structural representation of apoE isoforms in the context of lipoproteins and while interacting with Aβ, integrating the biochemical characterization of Core B, and facilitating investigations of isoform-specific functional effects conducted by Projects 2-5 and Core B-F.