Well-Defined Nanoparticles Formed by Hydrophobic Assembly of a Short and Polydisperse Random Terpolymer, Amphipol A8-35

Well-Defined Nanoparticles Formed by Hydrophobic Assembly of a Short and Polydisperse Random Terpolymer, Amphipol A8-35
Received August 17, 2005
In Final Form: November 29, 2005
Web Release Date: January 6, 2006
Yann Gohon, Fabrice Giusti, Carla Prata, Delphine Charvolin, Peter Timmins, Christine Ebel, Christophe Tribet,* and Jean-Luc Popot*
ACS Publications
Copyright © 2006 American Chemical Society
Laboratoire de Physicochimie Mol»culaire des Membranes Biologiques, UMR 7099, CNRS and Universit» Paris-7, Institut de Biologie Physico-Chimique, CNRS FRC 550, 13 rue Pierre et Marie Curie, F-75005 Paris, France, Laboratoire de Physico-Chimie des PolymÀres et des Milieux Dispers»s, CNRS UMR 7615, ESPCI, 10 rue Vauquelin, F-75005 Paris, France, Large Scale Structures Group, Institut Laue-Langevin, Avenue des Martyrs, B.P.156, F-38042 Grenoble Cedex 9, France, and Laboratoire de Biophysique Mol»culaire, Institut de Biologie Structurale, UMR 5075 CEA-CNRS-UJF, 41 rue Jules Horowitz, F-38027 Grenoble Cedex 01, France
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?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 ~40 kg?mol-1, a radius of gyration of ~2.4 nm, and a Stokes radius of ~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 (~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.
Amphiphilic polymers comprising both water-soluble and water-insoluble moieties tend to self-associate in water. This process, which is driven by the segregation of hydrophobic groups, forms the basis of a large variety of applications, including the replacement of high molecular weight viscosifiers,1 the dispersion of pigments and oils, the stabilization of proteins,2 and drug delivery.3,4 Structural investigations of the assemblies have been carried out mainly on diblock copolymers and telechelics, which comprise one hydrophobic and one hydrophilic segment, each of very low polydispersity. Despite specific properties arising from a significant contribution to free energy of the entropy of chain conformation, diblock copolymers are structurally similar to small surfactants, and they form micelles of low polydispersity.5,6 Other amphiphilic polymers with important applications include polysoaps and hydrophobically modified polymers (HMPs), which feature, respectively, regular and statistical distributions of hydrophobic side groups along a hydrophilic backbone. Of particular interest are the critical conditions for the onset of their self-association (pH, ionic strength, concentration, chemical composition, etc.)7 and the size of the hydrophobic clusters.8 The spatial organization of supramolecular associations is still a matter of debate, and there also remain many open questions about intrachain vs interchain associations,9 the size of hydrophobic microdomain(s), and the presence of multiple hydrophobic microdomains inside a single assembly (for reviews, see refs 7 and 10). Experimentally, the class of "closed" association was introduced to distinguish HMPs that can form clusters of constant size irrespective of concentration (e.g., pullulan-based HMPs11 and modified polyacrylamidosulfonates12), in contrast to most HMPs assemblies, whose size grows with concentration up to gelation.13-15
The formation of closed associations with a defined stoichiometry is a priori surprising for polydisperse HMPs grafted with statistical distributions of small hydrophobic groups. The absence of regularity in microstructure usually results in (i) a gradual transition between essentially free chains in dilute solution and mostly self-associated chains, with the fraction of hydrophobic clusters increasing slowly above the critical concentration of association,16 and (ii) the coexistence of hydrophobic clusters with different degrees of polarity.17,18 However, the existence of optimal compositions that would favor the formation of well-defined particles is not precluded. Fluorescence quenching studies have shown that solutions of polysoaps or HMPs can contain a large fraction of small hydrophobic microdomains, which allowed reliable measurements of the average aggregation number of the hydrophobic groups to be made.8,12,19,20 The prevalence of intra- over interchain associations causes a dramatic decrease of the reduced viscosity of some HMPs21 or polysoaps,22,23 with limited dependence on polymer concentration.
In the present article, we analyze the self-association properties of a derivative of poly(acrylic acid) (PAA) belonging to a family of HMPs called "amphipols" (APols), which was designed for membrane biology applications.24-28 APols are water-soluble, linear short-chain amphiphilic copolymers or terpolymers. Because each chain carries numerous hydrophobic groups, APols bind noncovalently but tenaciously to the hydrophobic transmembrane surface of integral membrane proteins (MPs).29-31 Thereby, they are able to keep them soluble in the absence of detergent.24,26,29-33 APols have many potential uses in structural and cell biology as well as in biotechnology, and they seem likely to hold some future in membrane biophysics and biochemistry (for reviews, see refs 27 and 28). They have recently been applied, for instance, to MP NMR studies31 and MP renaturation.33 However, the preparation and handling of MP/APol complexes demands a good understanding of the properties of the APols themselves, particularly since the size and dispersity of the complexes seem to critically depend on the solution behavior of the pure APols.30,32
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