Non-Ionic Amphiphilic Homopolymers: Synthesis, Solution Properties, and Biochemical Validation

Abstract

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, DOI: 10.1021/bi201862v).

Structural insights into biased G protein-coupled receptor signaling revealed by fluorescence spectroscopy

“…1B) revealed NAPols reconstituted V2R at around 75-80 kDa. These results are consistent with the molecular mass of a monomeric V2R (42 kDa) that would be in complex with at least three molecules of NAPol, on average (19). The enriched monomeric fraction was then used throughout the study.…”

“…Construction, expression, solubilization, and purification of the receptor mutants from Sf9 insect cells, as well as the labeling kinetics methods, are described in SI Text. Purified detergent-soluble receptors were incubated at 4°C in the presence of the amphipols at a 1:10 protein/NAPol weight ratio (19). After detergent removal with Bio-beads (Biorad), the sample was subjected to size exclusion chromatography (SI Text) to isolate the monomeric fraction of V2R.…”

“…In this study, we first demonstrate that purified V2R functionally reconstituted into a neutral amphipol (NAPol) (19) responds to ligands with the same efficacy profiles toward activation of purified Gs protein and arrestin-2 (β-arrestin-1) as in living cells. We then analyze the effects of biased ligands on the receptor structure.…”

Amphipol-Mediated Screening of Molecular Orthoses Specific for Membrane Protein Targets

“…To this end, a biotinylated non-ionic APol (BNAPol) was synthesized (Fig. 1b), derived from recently described homopolymeric NAPols Sharma et al 2012). An ITC titration of aRep bBAPol-F10, a binder that does interact with A8-35 (see above), did not reveal any interaction with NAPol (Fig.…”

“…1a) (Charvolin et al 2009), is a derivative of A8-35, a polyanionic, polyacrylate-based APol (Tribet et al 1996). More recently, a number of chemically different APols have been developed (reviewed in Zoonens and Popot 2014), among glucose-based nonionic amphipols ('NAPols'), whose complete absence of charges can be an asset under certain experimental circumstances Sharma et al 2012). As an alternative to BAPol, we have tested the use of a biotinylated NAPol ('BNAPol') ( Fig.…”