Non-Ionic Amphiphilic Homopolymers: Synthesis, Solution Properties, and Biochemical Validation.
Résumé
A novel type of nonionic amphipols for handling membrane proteins in detergent-free aqueous solutions has been obtained through free-radical homo-telomerization of an acrylamide-based monomer comprising a C 11 alkyl chain and two glucose moieties, using a thiol as transfer reagent. By controlling the thiol/monomer ratio, the number-average molecular weight of the polymers was varied from 8 to 63 kDa. Homopolymeric nonionic amphipols were found to be highly soluble in water and to self-organize, within a large concentration range, into small, compact particles of ∼6 nm diameter with a narrow size distribution, regardless of the molecular weight of the polymer. They proved able to trap and stabilize two test membrane proteins, bacteriorhodopsin from Halobium salinarum and the outer membrane protein X of Escherichia coli, under the form of small and well-defined complexes, whose size, composition, and shape were studied by aqueous size-exclusion chromatography, analytical ultracentrifugation, and small-angle neutron scattering. As shown in a companion paper, nonionic amphipols can be used for membrane protein folding, cell-free synthesis, and solution NMR studies (Bazzacco et al. 2012, Biochemistry, Amphiphilic molecules self-assemble in aqueous solvents because of the differential solvation properties of their hydrophilic and lipophilic groups. In water, detergents, a class of small surfactants, form micelles that can solubilize amphi-philic and lipophilic guest molecules. This property has proven highly useful in the study of membrane proteins (MPs), which are naturally water-insoluble due to the high hydrophobicity of their transmembrane surface. 1 However, the dissociating character of detergents, combined with the need to maintain an excess of them, tends to inactivate membrane proteins. This has prompted the development of milder surfactants, such as detergents with less disruptive structures, fluorinated surfac-tants, lipopeptides, or nanodiscs (for reviews, see refs 2−4). A particularly promising approach consists in generating multiple contacts between an amphiphilic polymer and the hydrophobic transmembrane surface of MPs, leading to the development of the so-called 'amphipols' (APols). 5 APols have proven very efficient at keeping MPs soluble in the absence of detergents, while stabilizing them biochemically. Their applications extend to MP folding, immobilization, structural studies by solution NMR and cryo-electron microscopy, and studies of ligand binding and the recruitment of effectors, as well as vaccination (reviewed in refs 4, 6, 7). The most extensively studied APol to date, A8−35, is an anionic polymer made of poly(acrylic acid) carrying multiple octyl chains. 5 Because its solubility depends on the carboxylate groups being charged, A8−35 suffers from certain limitations, among which are its insolubility in acidic buffers 8,9 and its sensitivity to calcium ions. 10 In addition , its charged character prevents the separation of MP/A8−35