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_Aβ42). 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_Aβ42's relevance in Alzheimer's disease (AD). However, βPFO_Aβ42 is formed under dodecyl phosphocholine (DPC) micelle conditions—intended to mimic the hydrophobic environment of membranes—which are dynamic. Consequently, dilution of the βPFO_Aβ42/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_Aβ42 from DPC micelles into another hydrophobic biomimetic membrane environment, that remains associated with βPFO_Aβ42 even under high dilution conditions, is a requisite for the validation of βPFO_Aβ42 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_Aβ42/DPC complex can only be trapped in non-ionic APols (NAPols). The characterization of the resulting βPFO_Aβ42/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_Aβ42 in AD.</p

Image1_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_Aβ42). 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_Aβ42's relevance in Alzheimer's disease (AD). However, βPFO_Aβ42 is formed under dodecyl phosphocholine (DPC) micelle conditions—intended to mimic the hydrophobic environment of membranes—which are dynamic. Consequently, dilution of the βPFO_Aβ42/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_Aβ42 from DPC micelles into another hydrophobic biomimetic membrane environment, that remains associated with βPFO_Aβ42 even under high dilution conditions, is a requisite for the validation of βPFO_Aβ42 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_Aβ42/DPC complex can only be trapped in non-ionic APols (NAPols). The characterization of the resulting βPFO_Aβ42/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_Aβ42 in AD.</p

Image2_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_Aβ42). 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_Aβ42's relevance in Alzheimer's disease (AD). However, βPFO_Aβ42 is formed under dodecyl phosphocholine (DPC) micelle conditions—intended to mimic the hydrophobic environment of membranes—which are dynamic. Consequently, dilution of the βPFO_Aβ42/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_Aβ42 from DPC micelles into another hydrophobic biomimetic membrane environment, that remains associated with βPFO_Aβ42 even under high dilution conditions, is a requisite for the validation of βPFO_Aβ42 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_Aβ42/DPC complex can only be trapped in non-ionic APols (NAPols). The characterization of the resulting βPFO_Aβ42/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_Aβ42 in AD.</p

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_Aβ42). 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_Aβ42's relevance in Alzheimer's disease (AD). However, βPFO_Aβ42 is formed under dodecyl phosphocholine (DPC) micelle conditions—intended to mimic the hydrophobic environment of membranes—which are dynamic. Consequently, dilution of the βPFO_Aβ42/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_Aβ42 from DPC micelles into another hydrophobic biomimetic membrane environment, that remains associated with βPFO_Aβ42 even under high dilution conditions, is a requisite for the validation of βPFO_Aβ42 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_Aβ42/DPC complex can only be trapped in non-ionic APols (NAPols). The characterization of the resulting βPFO_Aβ42/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_Aβ42 in AD.</p

Influence of Hydrophobic Groups Attached to Amphipathic Polymers on the Solubilization of Membrane Proteins along with Their Lipids

One of the biggest challenges in membrane protein (MP) research is to secure physiologically relevant structural and functional information after extracting MPs from their native membrane. Amphipathic polymers represent attractive alternatives to detergents for stabilizing MPs in aqueous solutions. The predominant polymers used in MP biochemistry and biophysics are amphipols (APols), one class of which, styrene maleic acid (SMA) copolymers and their derivatives, has proven particularly efficient at MP extraction. In order to examine the relationship between the chemical structure of the polymers and their ability to extract MPs from membranes, we have developed two novel classes of APols bearing either cycloalkane or aryl (aromatic) rings, named CyclAPols and ArylAPols, respectively. The effect on solubilization of such parameters as the density of hydrophobic groups, the number of carbon atoms and their arrangement in the hydrophobic moieties, as well as the charge density of the polymers was evaluated. The membrane-solubilizing efficiency of the SMAs, CyclAPols, and ArylAPols was compared using as models (i) two MPs, BmrA and a GFP-fused version of LacY, overexpressed in the inner membrane of Escherichia coli, and (ii) bacteriorhodopsin, naturally expressed in the purple membrane of Halobacterium salinarum. This analysis shows that, as compared to SMAs, the novel APols feature an improved efficiency at extracting MPs while preserving native protein–lipid interactions

A Step Closer to Membrane Protein Multiplexed Nanoarrays Using Biotin-Doped Polypyrrole

Whether for fundamental biological research or for diagnostic and drug discovery applications, protein micro- and nanoarrays are attractive technologies because of their low sample consumption, high-throughput, and multiplexing capabilities. However, the arraying platforms developed so far are still not able to handle membrane proteins, and specific methods to selectively immobilize these hydrophobic and fragile molecules are needed to understand their function and structural complexity. Here we integrate two technologies, electropolymerization and amphipols, to demonstrate the electrically addressable functionalization of micro- and nanosurfaces with membrane proteins. Gold surfaces are selectively modified by electrogeneration of a polymeric film in the presence of biotin, where avidin conjugates can then be selectively immobilized. The method is successfully applied to the preparation of protein-multiplexed arrays by sequential electropolymerization and biomolecular functionalization steps. The surface density of the proteins bound to the electrodes can be easily tuned by adjusting the amount of biotin deposited during electropolymerization. Amphipols are specially designed amphipathic polymers that provide a straightforward method to stabilize and add functionalities to membrane proteins. Exploiting the strong affinity of biotin for streptavidin, we anchor distinct membrane proteins onto different electrodes <i>via</i> a biotin-tagged amphipol. Antibody-recognition events demonstrate that the proteins are stably immobilized and that the electrodeposition of polypyrrole films bearing biotin units is compatible with the protein-binding activity. Since polypyrrole films show good conductivity properties, the platform described here is particularly well suited to prepare electronically transduced bionanosensors