Fabrice Giusti

Amphipols (APols) are short amphipathic polymers that can substitute for detergents to keep integral membrane proteins (MPs) water soluble. In this review, we discuss their structure and solution behavior; the way they associate with MPs; and the structure, dynamics, and solution properties of the resulting complexes. All MPs tested to date form water-soluble complexes with APols, and their biochemical stability is in general greatly improved compared with MPs in detergent solutions. The functionality and ligand-binding properties of APol-trapped MPs are reviewed, and the mechanisms by which APols stabilize MPs are discussed. Applications of APols include MP folding and cell-free synthesis, structural studies by NMR, electron microscopy and X-ray diffraction, APol-mediated immobilization of MPs onto solid supports, proteomics, delivery of MPs to preexisting membranes, and vaccine formulation.

One of the major obstacles to membrane protein (MP) structural studies is the destabilizing effect of detergents. Amphipols (APols) are short amphipathic polymers that can substitute for detergents to keep MPs water-soluble under mild conditions. In the present work, we have explored the feasibility of studying the structure of APol-complexed MPs by NMR. As a test MP, we chose the 171-residue transmembrane domain of outer MP A from Escherichia coli (tOmpA), whose x-ray and NMR structures in detergent are known. 2H,15N-labeled tOmpA was produced as inclusion bodies, refolded in detergent solution, trapped with APol A8-35, and the detergent removed by adsorption onto polystyrene beads. The resolution of transverse relaxation-optimized spectroscopy-heteronuclear single-quantum correlation spectra of tOmpA/A8-35 complexes was found to be close to that of the best spectra obtained in detergent solutions. The dispersion of chemical shifts indicated that the protein had regained its native fold and retained it during the exchange of surfactants. MP-APol interactions were mapped by substituting hydrogenated for deuterated A8-35. The resulting dipolar broadening of amide proton linewidths was found to be limited to the beta-barrel region of tOmpA, indicating that A8-35 binds specifically to the hydrophobic transmembrane surface of the protein. The potential of this approach to MP studies by solution NMR is discussed.

Amphipols are short amphilic polymers designed for applications in membrane biochemistry and biophysics and used, in particular, to stabilize membrane proteins in aqueous solutions. Amphipol A8-35 was obtained by modification of a short-chain parent polymer (poly(acrylic acid); PAA) with octyl- and isopropylamine, to yield an amphiphilic product with an average molar mass of 9-10 kg x mol(-1) (sodium salt form) and a polydispersity index of 2.0 to 3.1, depending on the source of PAA. The behavior of A8-35 in aqueous buffers was studied by size exclusion chromatography, static and dynamic light scattering, equilibrium and sedimentation velocity analytical ultracentrifugation, and small angle neutron scattering. Despite the variable length of the chains and the random distribution of hydrophobic groups along them, A8-35 self-organizes into well-defined assemblies. The data are best compatible with most of the polymer forming compact assemblies (particles) with a molar mass of approximately 40 kg x mol(-1), a radius of gyration of approximately 2.4 nm, and a Stokes radius of approximately 3.15 nm. Each particle contains, on average, four A8-35 macromolecules and 75-80 octyl chains. Neutron scattering reveals a sharp interface between the particles and water. A minor (approximately 0.1%) mass fraction of the material forms much larger aggregates, whose proportion may increase under certain conditions of preparation or handling, such as low pH. They can be removed by gel filtration.

Amphipols (APols) are short amphipathic polymers that can substitute for detergents to keep membrane proteins (MPs) water-soluble while stabilizing them biochemically. We have examined the factors that determine the size and dispersity of MP/APol complexes and studied the dynamics of the association, taking as a model system the transmembrane domain of Escherichia coli outer membrane protein A (tOmpA) trapped by A8-35, a polyacrylate-based APol. Molecular sieving indicates that the solution properties of the APol largely determine those of tOmpA/APol complexes. Achieving monodispersity depends on using amphipols that themselves form monodisperse particles, on working in neutral or basic solutions, and on the presence of free APols. In order to investigate the role of the latter, a fluorescently labeled version of A8-35 has been synthesized. Förster resonance energy transfer measurements show that extensive dilution of tOmpA/A8-35 particles into an APol-free medium does not entail any detectable desorption of A8-35, even after extended periods of time (hours-days). The fluorescent APol, on the other hand, readily exchanges for other surfactants, be they detergent or unlabeled APol. These findings are discussed in the contexts of sample optimization for MP solution studies and of APol-mediated MP functionalization.