Labeling and Functionalizing Amphipols for Biological Applications

Abstract

Amphipols (APols) are short amphipathic polymers developed as an alternative to detergents for handling membrane proteins (MPs) in aqueous solution. MPs are, as a rule, much more stable following trapping with APols than they are in detergent solutions. The best-characterized APol to date, called A8-35, is a mixture of short-chain sodium polyacrylates randomly derivatized with octylamine and isopropylamine. Its solution properties have been studied in detail, and it has been used extensively for biochemical and biophysical studies of MPs. One of the attractive characteristics of APols is that it is relatively easy to label them, isotopically or otherwise, without affecting their physical-chemical properties. Furthermore, several variously modified APols can be mixed, achieving multiple functionalization of MP/APol complexes in the easiest possible manner. Labeled or tagged APols are being used to study the solution properties of APols, their miscibility, their biodistribution upon injection into living organisms, their association with MPs and the composition, structure and dynamics of MP/APol complexes, examining the exchange of surfactants at the surface of MPs, labeling MPs to follow their distribution in fractionation experiments or to immobilize them, increasing the contrast between APols and solvent or MPs in biophysical experiments, improving NMR spectra, etc. Labeling or functionalization of APols can take various courses, each of which has its specific constraints and advantages regarding both synthesis and purification. The present review offers an overview of the various derivatives of A8-35 and its congeners that have been developed in our laboratory and discusses the pros and cons of various synthetic routes.

In Vivo Characterization of the Biodistribution Profile of Amphipol A8–35

“…It was purified by repeated cycles of precipitation at acidic pH and re-dissolution at basic pH, followed by SEC. As classically observed for APols grafted with small ligands (Zoonens et al 2007; Le Bon et al 2014), the solution behavior of FAPol AF647 , examined by SEC (Fig. 2), was identical to that of ungrafted A8–35, namely self-association into ~40-kDa globular particles (Gohon et al 2006), the purified polymer comprising ~1 fluorophore per particle.…”

“…Depending on the administration route, APol-associated MPs or drugs can be delivered in a long-term manner, eventually benefitting from natural reservoirs such as fat. One may also note that, because it is possible to functionalize APols with essentially any desirable molecule (Le Bon et al 2014), targeting MPs, peptides, or drugs to selected organs using APols tagged with organ-specific ligands is a realistic proposition.…”

Amphipols for Each Season

“…PC-APols and NAPols have also been tagged with biotin (Basit et al 2012; Ferrandez et al 2014). Chemical approaches to labeling and functionalizing APols are reviewed elsewhere in this issue (Le Bon et al 2014b). …”

“…provide more details and discussion about using APols for solution NMR studies of MPs (Planchard et al 2014), in synthetic biology (Della Pia et al 2014b), as a tool to manipulate biological membranes (Marie et al 2014), or for the study of specific classes of MPs (Huynh et al 2014; Mary et al 2014). A sixth one covers the important subject of the chemistry of APol labeling and functionalization (Le Bon et al 2014b). …”

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

“…Amphipols (APols) (Tribet et al 1996) are specially designed amphipathic polymers that have proven able to substitute to detergents for keeping water-soluble any MP tested so far, most of which are much more stable as complexes with APols than they are in detergent solutions (reviewed in Popot 2010;Popot et al 2011;. Because the association of APols to MPs is very strong and resist extensive dilution (Tribet et al 1997;Tribet et al 2009;Zoonens et al 2007), trapping a MP with a functionalized APol effectively functionalizes the protein (reviewed in, Le Bon et al 2014b;. Thus, an APol carrying a biotin tag can be used to attach MPs onto streptavidin-coated chips or beads (Charvolin et al 2009).…”