Langmuir 2006, 22, 1281-1290
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Well-Defined Nanoparticles Formed by Hydrophobic Assembly of a Short and Polydisperse Random Terpolymer, Amphipol A8-35 Yann Gohon,† Fabrice Giusti,† Carla Prata,‡,§ Delphine Charvolin,† Peter Timmins,| Christine Ebel,⊥ Christophe Tribet,*,‡ and Jean-Luc Popot*,† Laboratoire de Physicochimie Mole´ culaire des Membranes Biologiques, UMR 7099, CNRS and UniVersite´ 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 Polyme` res et des Milieux Disperse´ 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 Mole´ culaire, Institut de Biologie Structurale, UMR 5075 CEA-CNRS-UJF, 41 rue Jules Horowitz, F-38027 Grenoble Cedex 01, France ReceiVed August 17, 2005. In Final Form: NoVember 29, 2005 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.
Introduction 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 * Corresponding authors. E-mail:
[email protected] (C.T.);
[email protected] (J.-L.P.). † UMR 7099, CNRS and Universite ´ Paris-7. ‡ CNRS UMR 7615. § Present address: Chemistry Department, Metcalf Center for Science and Engineering, 590 Commonwealth Avenue, Boston, MA, 02215. | Institut Laue-Langevin. ⊥ UMR 5075 CEA-CNRS-UJF. (1) Alexandridis, P. Curr. Opin. Colloid Interface Sci. 1996, 1, 490-501. (2) Nomura, Y.; Ikeda, M.; Yamaguchi, N.; Aoyama, Y.; Akiyoshi, K. FEBS Lett. 2003, 553, 271-276. (3) Constancis, A.; Meyrueix, R.; Bryson, N.; Huille, S.; Grosselin, J.-M.; et al. J. Colloid Interface Sci. 1999, 217, 357-368. (4) Lukyanov, A. N.; Anatoly, N.; Torchilin, V. P. AdV. Drug DeliVery ReV. 2004, 56, 1273-1289. (5) Pedersen, J. S.; Svaneborg, C. Curr. Opin. Colloid Interface Sci. 2002, 7, 158-166. (6) Moffitt, M.; Khougaz, K.; Eisenberg, A. Acc. Chem. Res. 1996, 29, 95102.
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 (7) Laschewsky, A. AdV. Polym. Sci. 1995, 124, 1-86. (8) Petit-Agnely, F.; Iliopoulos, I.; Zana, R. Langmuir 2000, 16, 9921-9927. (9) Borisov, O. V.; Halperin, A. Macromolecules 1996, 29, 2612-2617. (10) Bromberg L. In Handbook of Surfaces and Interfaces of Materials; Nalwa, H. S., Ed.; Academic Press: New York, 2001; Vol. 4, Chapter 7, pp 369-404. (11) Kuroda, K.; Fujimoto, K.; Sunamoto, J.; Akiyoshi, K. Langmuir 2002, 18, 3780-3786. (12) Morishima, Y.; Nomura, S.; Ikeda, T.; Seki, M.; Kamachi, M. Macromolecules 1995, 28, 2874-2881. (13) Dobrynin, A. V.; Rubinstein, M. Macromolecules 2000, 33, 8098-8105. (14) Candau, F.; Selb, J. AdV. Coll. Interface Sci. 1999, 79, 149-172. (15) Hashidzume, A.; Noda, T.; Morishima, Y. ACS Symp. Ser 780; 2001; Chapter 2.
10.1021/la052243g CCC: $33.50 © 2006 American Chemical Society Published on Web 01/06/2006
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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 welldefined 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 intraover 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 Here, we analyze some important solution properties (size, molecular mass, dispersity as a function of concentration and pH) of the assemblies formed by the most widely used APol, A8-35. A8-35 is obtained by derivation with octyl- and isopropylamine of a short-chain PAA.24,34 According to the APol nomenclature used in ref 24, “A” stands for anionic, “8” refers to the average molar mass (see below), and “35” is the percentage (16) Petit, F.; Iliopoulos, I.; Audebert, R. Polymer 1998, 39, 751-753. (17) Petit-Agnely, F.; Iliopoulos, I. J. Phys. Chem. B 1999, 103, 4803-4808. (18) Furo, I.; Iliopoulos, I.; Stilbs, P. J. Phys. Chem. 2000, 104, 485-494. (19) Binana-Limbele, W.; Zana, R. Macromolecules 1990, 23, 2731-2739. (20) Olea, A. F.; Thomas, J. K. Macromolecules 1989, 22, 1165-1169. (21) Hu, Y.; Smith, G. L.; Richardson, M. F.; McCormick, C. L. Macromolecules 1997, 30, 3526-3537. (22) Heitz, C.; Pendharkar, S.; Prud’homme, R. K.; Kohn, J. Macromolecules 1999, 32, 6652-6657. (23) Qiu, Q.; Lou, A.; Somasundaran, P.; Pethica, B. A. Langmuir 2002, 18, 5921-5926. (24) Tribet, C.; Audebert, R.; Popot, J.-L. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 15047-50. (25) Prata, C.; Giusti, F.; Gohon, Y.; Pucci, B.; Popot, J.-L.; et al. Biopolymers 2001, 56, 77-84. (26) Gorzelle, B. M.; Hoffman, A. K.; Keyes, M. H.; Gray, D. N.; Ray, D. G.; et al. J. Am. Chem. Soc. 2002, 124, 11594-5. (27) Popot, J.-L.; Berry, E. A.; Charvolin, D.; Creuzenet, C.; Ebel, C.; et al. Cell. Mol. Life Sci. 2003, 60, 1559-74. (28) Sanders, C. R.; Hoffmann, A. K.; Gray, D. N.; Keyes, M. H.; Ellis, C. ChemBioChem 2004, 5, 423-426. (29) Tribet, C.; Audebert, R.; Popot, J.-L. Langmuir 1997, 13, 5570-76. (30) Zoonens, M. The`se de Doctorat d’Universite´, Universite´ Paris-6, Paris, 2004. (31) Zoonens, M.; Catoire, L. J.; Giusti, F.; Popot, J.-L. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 8893-8898. (32) Gohon, Y. The`se de Doctorat d’Universite´, Universite´ Paris-VI, Paris, 2002. (33) Pocanschi, C. L.; Dahmane, T.; Gohon, Y.; Apell, H.-J.; Kleinschmidt, J. H.; Popot, J.-L. Submitted for publication. (34) Gohon, Y.; Pavlov, G.; Timmins, P.; Tribet, C.; Popot, J.-L.; et al. Anal. Biochem. 2004, 334, 318-334.
Gohon et al. Scheme 1. Chemical Structure of Amphipol A8-35a
a From ref 24. Average molar masses and degrees of derivation for each of the batches of A8-35 used in the course of the present work are collected in Table 1; x, y, and z are respectively the molar fraction (in percents of units) of ungrafted carboxylate and of octylamide and isopropylamide side chains.
of free, ungrafted carboxylate groups.35 Under its sodium salt form, the final product has an actual average molar mass of 9-10 kg‚mol-1, depending on the average length of the PAA precursor.36 About 25% of the carboxylate groups are derived with octylamine and 40% with isopropylamine (Scheme 1 and Table 1). Deuterated derivatives of A8-35 have been synthesized in order to facilitate small angle neutron scattering (SANS), NMR, and analytical ultracentrifugation (AUC) studies of APols and MP/APol complexes.31,32,34 In a previous work, we have established the specific volume and ionization state of the hydrogenated and deuterated forms of A8-35 in aqueous solution.34 In the present one, A8-35 solutions have been studied by size exclusion chromatography (SEC), static and dynamic light scattering (SLS and DLS), AUC, and SANS. Discriminating between the formation of closed, well-defined self-assemblies and less organized or polydisperse aggregates was addressed in two ways: (i) by studying size distributions by DLS, AUC, and SEC, and (ii) by using SANS to gather information on the structure of the assemblies. We show that A8-35 self-organizes primarily into small, compact particles with a well-defined size. Materials and Methods Buffers. Buffer P7.1H was 100 mM sodium chloride, 20 mM (NaH2PO4-Na2HPO4), pH 7.1. Buffer P7.1D was obtained by lyophilisation of a known volume of buffer P7.1H and redissolution in the same volume of D2O. Buffer T8.5 was 100 mM sodium chloride, 20 mM Tris/Tris chlorhydrate pH 8.5. Buffer P6.8 was 100 mM NaCl, 20 mM sodium dihydrogenophosphate (NaH2PO4)-NaOH, pH 6.8. Buffer B9.2 was 100 mM sodium chloride, 20 mM boric acid-NaOH, pH 9.2. Buffers Tc7.5 and Tc8 were 100 mM sodium chloride, 20 mM Tricine-HCl, pH 7.5 and 8.0, respectively. Amphipol Synthesis. The synthesis protocol is detailed in the Supporting Information (SI). The composition of APol batches (Scheme 1 and Table 1) was determined by elemental analysis, acido-basic titration, and 1H and 13C NMR analysis in deuterated methanol as described in ref 34. “DAPol” and “HAPol” refer to PAA modified by grafting either perdeuterated or hydrogenated octylamine and isopropylamine, respectively. HAPol-1, DAPol-1, and DAPol-2 were synthesized using (hydrogenated) PAA from Aldrich (M ≈ 6500 g‚mol-1 under its sodium form), with a polydispersity index of 2.0, HAPol-2 and HAPol-3 using PAA from Acros (M ≈ 7200 g‚mol-1, polydispersity index 3.1). Amphipol Fractionation by SEC. Aqueous solutions of HAPol-2 at 25 g‚L-1 in buffer T8.5 contained numerous aggregates (see below), which could be separated by loading 2-mL samples on a 500-mL Superose 12 XK-26/100 column (Pharmacia) and eluting them with T8.5 buffer at room temperature (see Figure S1 in the SI). Five 10-mL fractions containing the smallest particles were pooled per run. The procedure was repeated to purify a total of 1 g of HAPol, which was then concentrated to 50 mL in a Millipore Alphacell stirred-cell with 10-kg‚mol-1 porosity (Filtron Technology) under (35) A8-35 is designated 5-25C8-40C3 in refs 37-39. (36) A8-35 was originally thus designated for the sake of consistency with the name of its 8 kg‚mol-1 precursor, A8-75, which carries no isopropyl groups.24
Well-Defined Amphipol A8-35 Nanoparticles
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Table 1. Compositiona of Different Batches of A8-35 and Stokes Radiib of the Small Particles composition A8-35 batch (lab. code) HAPol-1 (CT-961020H) HAPol-2 (FGHU3) HAPol-2′ (FGHU3frac) HAPol-3 (FGH20) DAPol-1 (CT-980225D) DAPol-2f (CP-990520D)
PAAc
origin
〈M〉 (g‚mol )
octylamine
isopropylamine
free carboxylate
impuritiese
RS
9000 10 000 10 000 10 000 9500 9500
28% 24% 25% 25% 27% 25%
41% 32% 34% 40% 44% 39%
31% 34% 35% 33% 29% 34%
n.d. 10% 6% 2% 3% 2%
3.15 polyd. 3.26 3.18 3.15 3.6
Aldrich Acros Acros Acros Aldrich Aldrich
d
-1
a Composition was determined by 1H and 13C NMR, acido-basic titration of carboxylic functions and elemental analysis as described in ref 34; errors are about (3%. “n.d.”: not detected. HAPol-2′ was obtained after fractionation of HAPol-2 by SEC (see text and Figure S1 in the SI).b RS is the Stokes radius measured by SEC after calibrating the column with soluble protein standards. “polyd.”: several peaks were detected upon SEC (see Figure S1 in Supporting Information). c Two different sources of sodium polyacrylate were used. d 〈M〉 is the average molar mass of one molecule of polymer, deduced from its composition and the average molar mass of the parent PAA (with 10% error), with sodium as the counterion. e Most likely dicyclohexylurea (DCU; see the SI). f Elemental analysis of lyophilized DAPol-2 suggested the co-lyophilisation of about 0.3 g salt per g of polymer.
2 atm prior to filtration through a 0.2-µm seringe filter (Durapore Millex-GV, VWR) and 4-h dialysis against water (Spectra/Por dialysis membrane, VWR, cutoff 6-8 kg‚mol-1). The resulting solution was freeze-dried to yield 0.5 g of purified polymer (HAPol2′). Analytical Size Exclusion Chromatography. An A ¨ kta Explorer 100 (Pharmacia) FPLC system equipped with a Superose 12 HR 10/30 column (23.6 mL; Pharmacia) and UV detection was used at either 4 °C or room temperature. Flow rate was 0.5 mL‚min-1. Solutions of APol or PAA (under its sodium form) were prepared at 100 g‚L-1 in water and diluted to 10 g‚L-1 into buffer P7.1H or T8.5 prior to loading 60-µL samples onto the column preequilibrated with the dilution buffer. Column calibration was done according to ref 40 using protein standards at 2-5 g‚L-1 in buffer P7.1H. The calibration curve (not shown) was fitted with a five-order polynomial according to ref 41. It was checked that multiple injections of APols had no effect on subsequent calibration runs. Static and Dynamic Light Scattering (SLS, DLS). For SLS and DLS, the freeze-dried polymer powder was dissolved in deionized water under gentle stirring for 24 h at room temperature, under N2, at concentrations of 2-40 g‚L-1. APol solutions were prepared daily, but our data shows that no significant modification occurs in pure water for several weeks. The polymer solution in water was mixed with an equal volume of 2× concentrated buffer (P6.8, Tc7.5, Tc8, or B9.2). The samples were filtered through a 0.22-µm syringe filter (Millex, Millipore USA). Measurements were carried out with an ALV-5000 multi-τ digital autocorrelator system employing a 3 W, 514.5 nm argon-ion laser (SP2020, Spectra Physics, CA) and an avalanche photodiode detector mounted on a rotating arm (PCS100, Malvern Instruments, England). Measurements were usually performed at 25 ( 1 °C and 90° scattering angle, with additional angledependence measurements in the range 140°-30°. Refractive index increments were measured in a differential refractometer (SpectraSystem RI-150, Thermo Finnigan, USA) with 1-5 g‚L-1 polymer solution in the corresponding buffer, with the following results: 0.119 g‚mL-1 for PAA in buffer P6.8, and 0.151 and 0.145 g‚mL-1 for HAPol-1 in buffers P6.8 and B9.2, respectively. The apparent weight average molar mass, Mapp, and virial coefficient A2 of the polymer in solution was obtained from Zimm extrapolations. In the absence of marked polymer aggregation, the radius of gyration was found to be too small to be determined (i.e., 30% w/w) in water and aqueous buffers. Slight differences of average molar mass and polydispersity of the parent PAA (see Materials & Methods) have no detectable incidence upon the solution behavior of the final product. On the other hand, it is critical to limit the extent of grafting, during the synthesis, of a byproduct of the coupling reaction, dicyclohexylurea (DCU; see Table 1 and the SI), which renders the polymer prone to aggregation (see below). Size Exclusion Chromatography. As shown in Figure 1, HAPol-1 and DAPol-1 migrated in T8.5 buffer (100 mM NaCl, 20 mM Tris/HCl, pH 8.5) as a single species, eluting slightly ahead of horseradish peroxidase (RS ) 3.0 nm). APol peaks were almost symmetrical, with half-height widths (HHWs) ∼1.35 mL, i.e., slightly larger than observed for proteins of comparable size, such as albumin or peroxidase (0.9-1.0 mL), which suggests some degree of polydispersity. Changing both the running buffer and that used to prepare the sample from T8.5 (pH 8.5) to P7.1H (pH 7.1) had no significant effect on the behavior of HAPol-1 (Figure 1). By reference to standard proteins, the position of the HAPol-1 and DAPol-1 peaks corresponds to a Stokes radius RS ) 3.15 ( 0.15 nm (Table 2). A very small amount of APol eluted in the void volume of the column, whereas some material trailed behind the main peak. The PAA precursor, on the other hand, formed a markedly broader zone (HHW ≈ 3.4 mL), eluting later than APols (see the SI, Figure S1). Because the apparent size of HAPol-1 and DAPol-1 is larger than that of their hydrophilic precursor and their elution peak is narrower, it seems likely that APols form supramolecular assemblies with a relatively narrow size distribution. As shown below, the mass of these objects indeed implies that each of them comprises several A8-35 molecules. Some early batches of A8-35 exhibited more complex elution patterns. HAPol-2, for instance, featured a highly polydisperse particle distribution: very large species eluted in the void volume of the column (RS > 9 nm), and a broad peak was observed between the void volume and the peak of small assemblies (see the SI, Figure S1). This behavior was characteristic of samples
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Figure 2. Dynamic light scattering analysis of the size distribution of HAPol-1 particles. Evolution of the size distribution of HAPol-1 particles upon incubation for 0-25 h in B9.2 buffer. All HAPol-1 concentrations were 10 g‚L-1.
with a high degree of artifactual grafting by DCU (typically >6%, see Table 1). The broad size distribution found in such batches suggests that some APol molecules, presumably because of their composition and/or a nonrandom distribution of hydrophobic side chains, induce the formation of larger assemblies. This hypothesis is supported by SEC experiments, in which the smallest HAPol-2 assemblies were separated from the rest of the preparation. The resulting sample (HAPol-2′) featured a very low dispersity, the width of its single peak (HHW ) 0.79 mL; see the SI, Figure S1) being significantly less than that observed upon fractionation of unprocessed HAPol-1 (Figure 1). Neither the dispersity of this sample nor that of larger HAPol-2 assemblies (also isolated by SEC) evolved after one month of storage at room temperature in water or buffer T8.5. The molar fraction of impurities in HAPol-2′ was found to be shifted, as compared to that of unfractionated HAPol-2, toward that of the other batches, falling from ∼10% to ∼6% (Table 1). This observation further supports the view that artifactual grafting by DCU promotes the aggregation of A8-35 molecules, presumably because it increases their hydrophobicity. Guidelines are provided in Supporting Information about how to avoid this problem. Static and Dynamic Light Scattering. SLS provided estimates of apparent molar masses, whereas DLS yielded information about Stokes radii. Both techniques are very sensitive to the presence of large particles, such as the aggregates observed by SEC. Table 2 summarizes the results obtained from SLS measurements performed less than 1 h after dilution of HAPol-1 samples to 3-20 g‚L-1. The average molar mass of HAPol-1 appeared close to 100 kg‚mol-1. The Rayleigh ratio was invariant over most of the q range, which indicates an average radius of gyration well below 15 nm. In keeping with SEC analysis (Figure S1), unmodified PAA appeared much smaller: ∼8 kg‚mol-1 at pH 9.2 (buffer B9.2), with a large uncertainty due to weak scattering. The presence of even a small fraction of large aggregates is troublesome for SLS and certainly shifts the observed molar mass toward values larger than the actual mass of the small APol assemblies (see below). The invariance of the observed mass with pH in the range 6.8-9.2 nevertheless reliably points to a fixed size-distribution of assemblies under these conditions. The second virial coefficient, A2, was positive and decreased upon lowering the pH (Table 2), which is qualitatively expected considering a moderate degree of Coulombic repulsion between HAPol-1 assemblies. We have shown elsewhere that, above pH 7, most of the free carboxylates carried by A8-35 are dissociated.34 A positive value of A2 points to the absence of significant hydrophobic attraction between APol assemblies.
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Figure 3. Small angle neutron scattering by DAPol-1 and HAPol1. (A) Effect of dilution of DAPol-1 in buffer P7.1H. (B) Normalized Guinier plots for hydrogenated A8-35 (HAPol-1) at different percentages of D2O in buffer P7.1. Dashed vertical lines define the region chosen (0.8 < QRg < 1.6) to determine the molar mass and Rg of the small particles. All samples were at 10 g‚L-1.
DLS measurements yield the intensity-weighted distribution of apparent Stokes radii (Figure 2). The distributions observed did not depend on the scattering angle to any significant extent (see the SI, Figure S2), which confirms the validity of the analysis in terms of diffusivity and corresponding apparent Stokes radius. A mode with remarkable low dispersity and small Stokes radius (RS ) 3.6 ( 1.5 nm) dominates the scattering at basic pH (Figure 2, buffer B9.2) and, for short incubation times, at lower pH as well (buffer P6.8; see the SI, Figure S3). The invariance of this mode in the concentration range 3-10 g‚L-1 validates the analysis in terms of hydrodynamic radius, with weak interparticle interactions. The intensity of scattered light is proportional to M and to the mass concentration. Based on the assumption of M ∝ RνS, with 2 < ν < 3, the ratio of the scattering by the small objects (RS ≈ 3.6 nm, 70% of the intensity) and larger scatterers (RS ) 40 ( 20 nm) implies that their mass ratio is g300. This conclusion is consistent with SEC analyses, which show that larger species comprise a negligible mass fraction of HAPol-1 (Figure 1). In basic solutions, the size distribution was stable over time (Figure 2). As discussed below, a markedly different behavior appeared when the pH was dropped below 7. Neutron Scattering. Small angle neutron scattering (SANS) is well adapted to studying dispersions of particles and to probing their internal structure, especially when isotopic labeling can be implemented. Two deuterated batches of A8-35 were synthesized (DAPol-1 and -2), in which both the isopropyl and the octyl side chains were perdeuterated, and a detailed SANS analysis of HAPol and DAPol solutions was carried out. In Figure 3A, Guinier plots of the intensity scattered by DAPol-1 at different concentrations (1.2-9.5 g‚L-1) have been normalized by the concentration. The good superimposition of
Gohon et al.
data points for all concentrations (but the highest) reflects the absence of significant interactions between APol assemblies, as well as insignificant dissociation of assemblies upon dilution. The slight decrease in forward scattered intensity observed at 9.5 g‚L-1 DAPol indicates the onset of interparticle repulsion. Scattering by the predominant species identified by both DLS and SEC, i.e., with RS between 3.1 and 3.6 nm, is expected to feature a linear dependence of log(I) vs Q2 down to Q ) 0.48 In keeping with DLS and SEC data, Guinier plots for HAPol-1 and DAPol-1 solutions indeed are linear in the region 0.0015 < Q2 < 0.0055 Å-2, consistent with the presence of a homogeneous population of small objects, whereas the abrupt upward deflection seen at low angles reflects that of significantly larger aggregates (Figure 3A,B). Below Q2 ≈ 0.001 Å-2, scattering is mostly due to large species with Rg > 10 nm. At larger Q (0.0015 < Q2 < 0.0055 Å-2), the contribution of the large species vanishes and scattering is dominated by the small one. The slope of the Guinier plot at high Q yields Rg ≈ 2.3 nm for the small objects for both HAPol-1 and DAPol-1, irrespective of polymer concentration (Figure 3A). Analysis at Varying Contrast. The two populations of small objects and large aggregates have the same scattering length density (ref 34 and the SI), making it possible to analyze scattering curves in terms of a bimodal size distribution of species with similar compositions (eq 3). The large objects can be estimated to represent a negligible weight fraction (eq 4), either 90% in mass of the material. The reduced sedimentation coefficients (s20,w) of the major species were 1.6 and 2.2 ( 0.1 S for HAPol-1 and DAPol-1, respectively. Consensus Model for Small APol Particles. Data from SEC, DLS, SANS, and AUC all indicate that A8-35 solutions are comprised of a majority of fairly homogeneous small objects along with a very low (∼0.1%) mass fraction of larger aggregates (RS > 9 nm according to SEC and SANS, ∼40 nm according to DLS). The small objects can be described as particles, given their globular and compact structure, as indicated by SANS. In the present section, we focus on integrating the ensemble of data collected on these small particles and deriving a consensus picture thereof. Table 2 summarizes the most relevant data collected, using six different techniques, on the two most thoroughly studied samples, HAPol-1 and DAPol-1. Stokes Radius. A constant value of RS ) 3.15 ( 0.15 nm was obtained by SEC (using soluble proteins as standards) for all HAPol and DAPol batches that were not polydisperse (Tables 1 and 2). Similar results were obtained in buffers P7.1H and T8.5. This value can be compared with that deduced from SANS data if one assumes, as a first (rough) approximation, a homogeneous population of spherical particles. The Stokes radius deduced from SANS data, Rs ) R°g × (5/3)1/2 ) 3.1 ( 0.25 nm for both HAPol-1 and DAPol-1 (Table 2), perfectly matches that from SEC. This excellent agreement points to the small APol particles being compact and globular, which, as discussed above, is also indicated by the rapid drop of I(Q) at high angles observed in SANS experiments. The low Rg/RS ratio of 0.76 ( 0.10 points to a limited ellipticity, with an axial ratio (a/b) comprised between 0.45 and 2. DLS measurements provide a Stokes radius of 3.6 ( 1.5 nm; this slightly larger value likely reflects some degree of polydispersity, in keeping with the result of SEC analyses. Barring the case of HAPol-2, a sample with a large degree of artifactual DCU grafting, RS values determined by SEC were remarkably constant from one batch of A8-35 to the next (Table 1). This is all the more significant given the variations in average mass and polydispersity entailed by the use of different PAA sources. Polydispersity of the Small Particles. The peaks observed upon SEC analysis of unfractionated APol batches such as HAPol-1 and DAPol-1 were slightly broader than those of the soluble proteins used as standards, which again suggests that the small particles are not perfectly monodisperse. This is confirmed by the AUC experiments. Molar Mass. Three approaches were resorted to, namely SANS and equilibrium and sedimentation AUC: (1) Extrapolation to Q ) 0 of the linear part of SANS Guinier plots yields molar masses of 46 and 34 ( 1.5 kg‚mol-1for the small particles present in HAPol-1 and DAPol-1, respectively (Table 2). (2) Another set of values, 28 and 42 kg‚mol-1 for HAPol-1 and DAPol-1, respectively (Table 2), can be derived from sedimentation equilibrium AUC. These values must be considered somewhat critically, however, because small particles with different masses partially separate from each other during the experiment. (3) Sedimentation velocity experiments reveal a major form of small particles, representing 70-90% of the mass of the sample, and one or several minor ones, migrating faster (Figure 6). The ratio s20,w/(1 - φ′F), which is proportional to M/RS, is the same for the major species of HAPol-1 and DAPol-1, suggesting that their dimensions and molar masses in solution are nearly identical
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Figure 5. Sedimentation equilibrium profiles of HAPol-1 and DAPol-1. Experimental profiles (absorbance at 240 nm) are shown in panels B (HAPol-1) and D (DAPol-1) at 12 000 rpm (b) and 18 000 rpm (O). Both samples were at 0.5 g‚L-1 in buffer P7.1H, 5 °C. The solid and dotted lines in panels B and D are fits considering a single noninteracting species and no baseline correction. They yield buoyancy molar masses of 3300 and 3000 g‚mol-1 (HAPol) and 7400 and 7000 g‚mol-1 (DAPol) at 12 000 and 18 000 rpm, respectively. Panels A and C show the residuals between experimental data and models. They are not homogeneously distributed around a null value, indicating that the samples are slightly heterogeneous.
Figure 7. Effect on neutron scattering by DAPol-2 (10 g‚L-1) of extended incubation in either buffer B9.2 (pH 9.2) or buffer P6.8 (pH 6.8). Figure 6. Sedimentation velocity analyses of HAPol-1 and DAPol1. (A) Dots: a selection of experimental sedimentation velocity profiles obtained at 5 °C and 60 000 rpm for HAPol-1 in buffer P7.1H. Absorbance at 230 nm was recorded every 28 min for a total of 4.5 h. Solid lines: fits using the program SEDFIT,47 assuming a continuous distribution of particles. (B) Residuals. (C) Corresponding c(s20,w) distributions for HAPol-1 (solid line) and DAPol-1 (dotted line), obtained under identical experimental conditions.
(Table 2). Svedberg’s equation (eq 6) can be used to derive a molar mass by combining the sedimentation coefficient s of this major species with φ′ and RS(SEC). Because the RS and s values used refer to slightly different subpopulations of particles, the molar masses thus obtained, 42 and 44 ( 5 kg‚mol-1 for HAPol-1 and DAPol-1, respectively, are expected to be slightly underestimated. They are nevertheless close to those derived from SANS and from equilibrium sedimentation experiments (Table 2). The molar mass obtained by averaging the results from the two most reliable techniques, SANS and sedimentation AUC, is ∼40 ( 5 kg‚mol-1, which appears to constitute a reasonable overall consensus value. This corresponds to ∼75-80 octyl groups per small particle, and an average of ∼4 APol chains. The relatively narrow size distribution of the particles suggests that the number of octyl chains must be relatively constant from one particle to the next. On the other hand, given the chain length
polydispersity, the number of macromolecules per particle must fluctuate. The size of the particles, given their mass and the specific volume of the dry polymer,34 suggests a relatively high degree of hydratation (Table 2; see the SI). Kinetics of Formation and Break-up of Large Aggregates. The evolution of A8-35 solutions as a function of the time elapsed since solubilization was explored using DLS and SANS. We consider here SANS results obtained with DAPol-2 and give additional data from DLS as SI. For freshly prepared solutions of DAPol-2, SANS data at pH 6.8 (buffer P6.8) or pH 9.2 (buffer B9.2) were qualitatively similar to that measured for DAPol-1 in buffer P7.1H (Figure 7). At pH 9.2, the linear analysis of Guinier plots beyond Q2 ) 0.0004 Å-2 yielded M ≈ 27.5 kg‚mol-1 and Rg ) 1.9 ( 0.1 nm for the small particles. At this pH, the evolution over time remained, after 36 h at room temperature, within statistical fluctuations through most of the Q range: only a very limited increase ( 7.0), nearly complete disappearance of the hydrophobic clusters present at low pH, as long as the salt concentrations is 50% sulfonate units.12 An important difference between these earlier works and the present one is the relatively short length of A8-35, and, therefore, the smaller number of hydrophobic groups carried by any single molecule (on average ∼20 in A8-35, vs ∼400 and ∼1000 in the two examples quoted). If a minimal number of ∼50-100 hydrophobic groups is needed to stabilize a hydrophobic cluster,8,17 this is bound to shift the equilibrium toward multimolecular assemblies. The phenomenon of HMP self-assembly is usually described as markedly less cooperative than surfactant micellization, principally because of (i) constraints due to the polymer backbone and (ii) the molecular heterogeneity of the macromolecules generally used.9,16 An increase of the average molar mass of assemblies as a function of polymer concentration has been observed in some cases12,50 and tentatively attributed to changes of the equilibrium concentration of free (unaggregated) hydrophobic groups. Well-defined aggregation numbers (Nagg, referring to the number of polymer chains in one micelle-like structure) are hardly evoked in the literature, except for single-chain collapse in dilute solution (Nagg ) 1). In one work, oligomers having a low polydispersity (3-5 hydrophobic groups per chain) were reported, on the basis of SLS data, to self-associate with apparent Nagg ≈ 4, though with an increase of apparent molar mass with polymer concentration.22 It has been argued that such data may actually reflect the coexistence of single molecules with a minor mass fraction of much larger aggregates.22,53 Such is definitely not the case for A8-35, since DLS, SEC, SANS, and AUC data all indicate that an overwhelming majority of the material is organized into well-defined paucimolecular assemblies, whose size and prevalence do not depend on polymer concentration. Neither the variable length of the chains nor the random distribution of octyl side groups hampers the assembly of welldefined particles. Both SEC and AUC data however indicate that those are not perfectly monodisperse. Particle polydispersity is more likely due to individual differences between molecules in the batch rather than to metastability or a broad equilibrium distribution of energetically nearly-equivalent sizes around an optimal one, based on the following two arguments: (i) fractions selected by SEC from a batch yielding a broad zone will yield a narrow one upon being fractionated again (SI, Figure S1, and unpublished observations);54 and (ii) exchange of molecules
(52) McCormick, C. L.; Shalaby, W.; Butler, G. B. Water soluble polymers: synthesis, solution properties and applications; American Chemical Society: Washington, DC, 1991; Vol. 467.
(53) Sauvage, E.; Plucktaveesak, N.; Colby, R. H.; Amos, D. A.; Antalek, B.; Schroeder K. M.; Tan J. S. J. Polym. Sci. Part B: Polym. Phys. 2004, 42, 35843597.
Discussion
1290 Langmuir, Vol. 22, No. 3, 2006
between small A8-35 particles is likely to be fast on the timescale of these experiments.57 It does not seem that the variable length of individual molecules be a critical factor in generating particle polydispersity, since batches of A8-35 synthesized from precursor PAAs with either a broad (3.1) or a very narrow (1.3) polydispersity index yielded particles with similar size distributions (C. T. & F. G., unpublished data). The hydrophobic/ hydrophilic balance of individual molecules and, possibly, the degree of randomness of octyl chain distribution are more likely to determine the size of the resultant particles. On average, small A8-35 particles comprise ∼4 macromolecules, bearing a total of 75-80 octyl chains. The latter number is close to that of octyl chains in the average octylglucoside (Nagg ≈ 82) or C8E5 (Nagg ≈ 90) micelle, two detergents frequently used in biochemistry.58 It is only slightly larger than the average number of octyl chains per hydrophobic cluster (∼50) found, using fluorescence techniques, for long-chain hydrophobically modified PAAs.8 This observation tends to suggest that A8-35 particles organize around a hydrophobic core whose size is similar to that of detergent micelles, a conclusion consistent with the variation of the Rg of deuterated A8-35 particles as a function of contrast in SANS experiments. The present data are of interest on several grounds. From the physical chemical point of view, APols were developed with the aim of providing a dense, firmly bound, and, if possible, tightly packed layer that would insulate the highly hydrophobic transmembrane region of membrane proteins from aqueous solutions. This objective dictated the choice of a relatively short, highly hydrophilic backbone densely grafted with short hydrophobic chains.24 The present study shows that these requirements result in molecules featuring an original solution behavior: APols, or at least A8-35, self-organize into well-defined, close to spherical micelle-like particles, in a concentration-independent manner that betrays large differences of standard free energy between (54) See also Figure 10 of ref 55; in these earlier experiments, however, the behavior of A8-35 particles was complicated by the aggregative effect of Ca2+ ions (see ref 56). (55) Champeil, P.; Menguy, T.; Tribet, C.; Popot, J.-L.; le Maire, M. J. Biol. Chem. 2000, 275, 18623-18637. (56) Picard, M.; Dahmane, T.; Garrigos, M.; Gauron, C.; Giusti, F.; et al. Biochemistry, in press. (57) Fluorescence resonance energy transfer (FRET) experiments (30) show that the exchange of molecules between free A8-35 assemblies and MP/A8-35 complexes is a relatively rapid phenomenon, occurring over tens of minutes to hours, depending on the ionic strength. (58) le Maire, M.; Champeil, P.; Møller, J. V. Biochim. Biophys. Acta 2000, 1508, 86-111.
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the isolated chain, the paucimolecular micelle, and larger types of assemblies. This is likely due to individual molecules carrying too few octyl chains to form by themselves large enough hydrophobic cores. The small particles have a size commensurate to that of small soluble proteins, and they are well suited to being studied using techniques developed by biophysicists and seldom used for polymer research, such as, in the present work, AUC. They provide a novel, promising model for studying the structure and dynamics of HMP assemblies. As regards biological applications, the present work opens on a number of interesting perspectives. First, it gives the biologist working with MP/APol complexes useful practical information about the behavior of free APols. To take a single example, it is clear from the present data that SEC is not well suited to separating complexes between small MPs and APols (e.g., tOmpA/A8-35 complexes, RS ≈ 5 nm,31 from free A8-35 (RS ≈ 3 nm)), whereas such a fractionation is straightforward for larger MP/APol complexes. Second, we show elsewhere that the solution behavior of a given APol or batch thereof is a fair predictor of that of the complexes it forms with MPs.30,32 Studying the behavior of free APols therefore is a worthwhile investment when optimizing conditions for studying APol-trapped MPs (see, e.g., ref 31). The very small fraction of large scatterers present in unfractionated batches of A8-35 poses no problem for biological applications, since they do not associate with membrane proteins,32 do not interfere with most biophysical measurements, and can be easily removed, if need be, by either SEC fractionation or ultracentrifugation. Acknowledgment. Particular thanks are due to D.M. Engelman for suggestions and advice about the use of SANS and for his participation in several of these experiments, to P. Herve´ for APol syntheses, and to M. Zoonens and G. Zaccaı¨ for discussions and comments on the manuscript. This work was supported by the CNRS, Universite´ Paris-7, the CEA, Universite´ Joseph Fourier, and grants to J.-L.P. from the CNRS interdisciplinary program Physique et Chimie du ViVant, the EU (BIO4C.T.98-0269), and HFSPO (RG00223/2000-M). Supporting Information Available: Further details on A8-35 synthesis and analyses (see main text). This material is available free of charge via the Internet at http://pubs.acs.org. LA052243G
