Image3_Stabilization of a Membrane-Associated Amyloid-β Oligomer for Its Validation in Alzheimer's Disease.TIF

<p>We have recently reported on the preparation of a membrane-associated β-barrel Pore-Forming Aβ42 Oligomer (βPFO<sub>Aβ42</sub>). It corresponds to a stable and homogeneous Aβ42 oligomer that inserts into lipid bilayers as a well-defined pore and adopts a specific structure with characteristics of a β-barrel arrangement. As a follow-up of this work, we aim to establish βPFO<sub>Aβ42</sub>'s relevance in Alzheimer's disease (AD). However, βPFO<sub>Aβ42</sub> is formed under dodecyl phosphocholine (DPC) micelle conditions—intended to mimic the hydrophobic environment of membranes—which are dynamic. Consequently, dilution of the βPFO<sub>Aβ42</sub>/DPC complex in a detergent-free buffer leads to dispersion of the DPC molecules from the oligomer surface, leaving the oligomer without the hydrophobic micelle belt that stabilizes it. Since dilution is required for any biological test, transfer of βPFO<sub>Aβ42</sub> from DPC micelles into another hydrophobic biomimetic membrane environment, that remains associated with βPFO<sub>Aβ42</sub> even under high dilution conditions, is a requisite for the validation of βPFO<sub>Aβ42</sub> in AD. Here we describe conditions for exchanging DPC micelles with amphipols (APols), which are amphipathic polymers designed to stabilize membrane proteins in aqueous solutions. APols bind in an irreversible but non-covalent manner to the hydrophobic surface of membrane proteins preserving their structure even under extreme dilution conditions. We tested three types of APols with distinct physical-chemical properties and found that the βPFO<sub>Aβ42</sub>/DPC complex can only be trapped in non-ionic APols (NAPols). The characterization of the resulting βPFO<sub>Aβ42</sub>/NAPol complex by biochemical tools and structural biology techniques allowed us to establish that the oligomer structure is maintained even under high dilution. Based on these findings, this work constitutes a first step towards the in vivo validation of βPFO<sub>Aβ42</sub> in AD.</p>