Article pubs.acs.org/Langmuir
Non-Ionic Amphiphilic Homopolymers: Synthesis, Solution Properties, and Biochemical Validation K. Shivaji Sharma,†,¶ Grégory Durand,*,†,‡ Frank Gabel,§,∥,⊥ Paola Bazzacco,# Christel Le Bon,# Emmanuelle Billon-Denis,# Laurent J. Catoire,# Jean-Luc Popot,*,# Christine Ebel,*,§,∥,⊥ and Bernard Pucci†,‡ †
Université d’Avignon et des Pays de Vaucluse, Equipe Chimie Bioorganique et Systèmes Amphiphiles, 33 rue Louis Pasteur, F-84000 Avignon, France ‡ Unité Mixte de Recherche 5247, Centre National de la Recherche Scientifique and Universités de Montpellier 1&2, Institut des Biomolécules Max Mousseron, Faculté de Pharmacie, 15 avenue Charles Flahault, F-34093 Montpellier Cedex 05, France § CEA, Institut de Biologie Structurale Jean-Pierre Ebel, 41 rue Jules Horowitz, F-38027, Grenoble, France ∥ CNRS, Institut de Biologie Structurale Jean-Pierre Ebel, Grenoble, France ⊥ Université Joseph Fourier − Grenoble 1, Institut de Biologie Structurale Jean-Pierre Ebel, Grenoble, France # Unité Mixte de Recherche 7099, Centre National de la Recherche Scientifique and Université Paris 7, Institut de Biologie Physico-Chimique, 13 rue Pierre-et-Marie Curie, F-75005 Paris, France S Supporting Information *
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 C11 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).
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the so-called ‘amphipols’ (APols).5 APols have proven very efficient at keeping MPs soluble in the absence of detergents, while stabilizing them biochemically. Their applications extend to MP folding, immobilization, structural studies by solution NMR and cryo-electron microscopy, and studies of ligand binding and the recruitment of effectors, as well as vaccination (reviewed in refs 4, 6, 7). The most extensively studied APol to date, A8−35, is an anionic polymer made of poly(acrylic acid) carrying multiple octyl chains.5 Because its solubility depends on the carboxylate groups being charged, A8−35 suffers from certain limitations, among which are its insolubility in acidic buffers8,9 and its sensitivity to calcium ions.10 In addition, its charged character prevents the separation of MP/A8−35
INTRODUCTION Amphiphilic molecules self-assemble in aqueous solvents because of the differential solvation properties of their hydrophilic and lipophilic groups. In water, detergents, a class of small surfactants, form micelles that can solubilize amphiphilic and lipophilic guest molecules. This property has proven highly useful in the study of membrane proteins (MPs), which are naturally water-insoluble due to the high hydrophobicity of their transmembrane surface.1 However, the dissociating character of detergents, combined with the need to maintain an excess of them, tends to inactivate membrane proteins. This has prompted the development of milder surfactants, such as detergents with less disruptive structures, fluorinated surfactants, lipopeptides, or nanodiscs (for reviews, see refs 2−4). A particularly promising approach consists in generating multiple contacts between an amphiphilic polymer and the hydrophobic transmembrane surface of MPs, leading to the development of © 2012 American Chemical Society
Received: December 20, 2011 Revised: February 2, 2012 Published: February 2, 2012 4625
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Scheme 1. Synthetic Strategy for the Preparation of Non-Ionic Amphipolsa
a
Left, co-telomers.15 Center, hydrophilic homo-telomers randomly grafted with hydrophobic chains.16 Right, amphiphilic homo-telomers (present work).
complexes by such techniques as ion exchange chromatography or isoelectric focusing. These constraints have prompted the development of alternative, chemically different APols, including zwitterionic APols,11,12 sulfonated APols (SAPols),13 and nonionic amphipols (NAPols).14−16 The first NAPols to be synthesized and validated were short copolymers obtained by radical co-telomerization of hydrophilic and lipophilic monomers derived from tris(hydroxymethyl)acrylamidomethane (THAM), in the presence of a transfer reagent. The water solubility of these polymers was rather poor, which limited their usefulness in biochemistry, but some of them were shown to be able to keep model MPs water-soluble and in their native state in the absence of detergents, providing a first proof of principle.14 More recently, the properties of glucose-based NAPols have been investigated. Two series of heteropolymers were generated, one by co-telomerization of hydrophilic and amphiphilic glucose-based monomers,15 the other by homo-telomerization of a hydrophilic glucose-based monomer followed by hydrophobization of the resulting homopolymer,16 yielding similar structures by very different routes (Scheme 1, left and center). The resulting polymers feature high water-solubility and surface activity, as well as the ability to self-organize into small and compact particles of well-defined size and to trap MPs and keep them water-soluble.16 However, none of the two synthetic routes allowed the preparation of large quantities of NAPols with high batch-to-batch reproducibility, a requirement for carrying out extended biochemical and biophysical investigations. The present article describes the development of a simpler and more convenient synthetic route
to amphiphilic polymers with related structures and validates them as efficient APols. Molecular designs based on amphiphilic homopolymers in which both hydrophilic and hydrophobic moieties are incorporated within the same monomer unit have been recently developed, leading to polymers with unique self-assembling properties and applications such as separation, catalysis, and sensing.17−19 In the present work, we describe the synthesis and properties of nonionic homopolymeric amphipols (NAhPols), in which each repeat unit is by itself an amphiphile (Scheme 1, right). NAhPols were obtained by free-radical telomerization, in the presence of a transfer reagent, of an acrylamide-based monomer comprising a C11 alkyl chain and two glucose moieties.20 The aqueous behavior of NAhPols was studied by surface tension measurements, dynamic light scattering (DLS), aqueous size exclusion chromatography (ASEC), analytical ultracentrifugation (AUC), and small-angle neutron scattering (SANS). Their potential for stabilizing MPs in aqueous solutions was studied using two model MPs, one whose transmembrane domain comprises seven α-helices and the cofactor retinal, bacteriorhodopsin (BR) from Halobium salinarum, and one that is made up of an eight-strand transmembrane β-barrel, Escherichia coli’s outer membrane protein X (OmpX). We show that NAhPols trap and stabilize both proteins in the form of small, well-defined complexes − thus offering the first example of homopolymeric APols. Perspectives for the applications of NA h Pols in MP biochemistry and biophysics are discussed. 4626
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curve of the absorbance vs. the molar concentration of TR was first established using standard solutions at 10−580 μM in CH2Cl2/MeOH (1:1 v/v). A precise weight of protected NAhPol (p-NAhPol) was dissolved in CH2Cl2/MeOH, defining the weight concentration of polymer in g·L−1. The value of the UV absorbance provided the molar concentration of the transfer reagent [TR] (mol.L−1) in the solution. M̅ n was then calculated by the following equation:
MATERIALS AND METHODS
Materials, general procedures and instrumentation for the synthesis are given in the Supporting Information. Synthesis of NAhPols (Scheme 2). The general procedure taking NA-29 as an example is as follows. The letters NA stand for ″nonionic
Scheme 2. Synthetic Pathway for Non-Ionic Amphiphilic Homopolymers (NAhPols)c
M̅ n = [p‐NAhPols]/[TR]
(1)
The DPn of the protected NAhPol was determined through the following equation: M̅ n = MTR + [(Mp M) × n]
(2)
where MTR is the molecular mass of the transfer reagent (MTR = 522 g·mol−1), MpM that of protected monomer pM (MpM = 1032 g·mol−1), and n the total number of monomers. The number-average degree of polymerization DPn is then obtained as DPn = n + 1
(3)
The number-average molecular weight of protected NAhPol was also obtained by 1H NMR calibration, by comparing the integral area of protons from the phenyl groups at 7.4−8.1 ppm with those of the NH vicinal methylene of each protected monomer constituting the polymer backbone. Deprotection of NAhPol. Protected NAhPol (2.0 g, 1.91 mmol) was dissolved in dry methanol (50 mL) under argon atmosphere. A catalytic amount of sodium methoxide was added and the mixture was stirred overnight at room temperature. The reaction solution was neutralized by addition of a spatula of acidic resin (IRC 50) (∼pH 8) and the solution was shaken for 15 min. The solution was filtered off, and then concentrated under vacuum. The crude polymer was dissolved in methanol (20 mL) and precipitated into cold ether (250 mL). The precipitate was filtered off and the solvent was evaporated under vacuum. Finally, the resulting NAhPol was solubilized in Milli-Q water at 10 g·L−1, filtered through a 0.45 μm PVDF syringe filter, and then subjected to dialysis against Milli-Q water for 24 h under continuous stirring using dialysis membrane tubing (MWCO 6−8 kDa). The resulting dialyzed solution was freeze-dried to give the NAhPol NA29 as a white powder (1.02 g, 78%). 1H NMR (250 MHz, DMSO-d6) δ (ppm) 7.1 (m, −NH), 5.2−4.8 (glucose unit), 4.6−3.4 (glucose unit, −CH2−O), 3.2 (m, −NH−CH2), 1.9−1.2 (CH2−CH of the polymer chain, CH2 of the alkyl chain), 0.8 (t, −CH3 of the alkyl chain). Deprotection of Monomer pM. The synthetic route was essentially the same as for the deprotection of NAhPol. The reaction solution was neutralized by addition of a spatula of acidic resin (IRC 50) (∼pH 8) and the solution was shaken for 15 min. The resulting deprotected monomer dM was solubilized in Milli-Q water and the solution was filtered through a 0.45 μm filter and subsequently freezedried to give the deprotected monomer N-1,1-di[(O-β-D-glucopyranosyl)oxymethyl]-1-[(undecylcarbamoyloxymethyl)-methyl)]acrylamide as a white powder in quantitative yield. 1H NMR (250 MHz, DMSO-d6) δ (ppm) 7.58 (s, 1H), 7.14 (t, J = 5.7 Hz, 1H), 6.33 (dd, J = 11.0 and 16.8 Hz, 1H), 6.05 (dd, J = 2.2 and 16.8 Hz, 1H), 5.64 (d, J = 11.0 Hz, 1H), 5.2−4.8 (m, 6H), 4.6−2.8 (m, 24H), 1.5− 1.1 (m, 18H), 0.87 (t, J = 6.5 Hz, 3H). 13C NMR (66.86 MHz, CD3OD) δ (ppm) 166.8, 157.4 (CO), 131.3 (CH), 125.7 (CH2), 103.5, 103.4, 76.6, 73.6, 70.1 (CH), 67.8, 67.7, 61.4 (CH2), 59.5 (C) 40.5, 31.7, 29.5, 29.3, 29.1, 26.5, 22.3 (CH2), 14.5 (CH3). HRMS (ESI+) calculated for C31H57N2O15 ([M + H]+) 697.3759, found 697.3748. Surface Tension Measurements. The surface activity of NAhPols and deprotected monomer dM in solution at the air−water interface was determined by the Wilhelmy plate technique using a Krüss K-100 (Krüss, Germany) tensiometer at 22 ± 1 °C. The polymer solution was prepared 12−24 h prior to measurement using Milli-Q water. Twenty milliliters of polymer solution were taken in a glass trough and the surface tension was determined by dilution. Other conditions were as reported elsewhere.21 Dynamic Light Scattering. The hydrodynamic particle size distribution and polydispersity of NAhPols and deprotected monomer
ca
Described in ref 15. bDescribed in ref 20. Reagents and conditions: (a) AIBN (0.5 equiv), THF at 66 °C or CH3CN at 82 °C, argon, 8−24 h, ∼30−60%; (b) MeONa, MeOH, pH 8−9, room temperature, argon, 12 h, ∼75% for NAhPols, quantitative yield for deprotected monomer dM. amphiphilic homopolymer″, followed by the number-average molecular weight expressed in kDa. Thus, “NA29” denotes a NAhPol with a number-average molecular weight M̅ n ≈ 29 kDa. Protected monomer pM20 (1.0 g, 0.97 mmol, 40.0 equiv) was dissolved in THF (15 mL). The solution was degassed by bubbling with argon for 20 min and then heated to reflux still under argon bubbling. The transfer reagent TR15 (12.62 mg, 0.024 mmol, 1.0 equiv) and AIBN (1.98 mg, 0.012 mmol, 0.5 equiv) dissolved in THF (1 mL) were then simultaneously added through a microsyringe. The reaction mixture was stirred at reflux until complete consumption of protected monomer (∼18 h). The reaction mixture was concentrated under vacuum, and the crude polymer was purified by gravity size-exclusion chromatography on Sephadex LH-20 resin eluting with a 1:1 (v/v) MeOH/CH2Cl2 mixture. The solvent was evaporated under vacuum to give the protected NAhPol as a white powder (0.524 g, 52%). Four sets of the above reaction were run and combined to give 2.07 g of NAhPol with an average 51% yield on the four reactions. 1H NMR (250 MHz, CDCl3) δ (ppm) 8.1−7.4 (m, C6H5 from TR), 6.6 (m, −NH), 5.3−4.8 (glucose unit: H4, H2, H3), 4.6−3.6 (glucose unit: H1, H6, H6′, H5, −CH2−O), 3.1 (m, −NH−CH2), 2.4−2.1 (bs, −OCOCH3), 1.9−1.3 (CH2−CH of the polymer chain, CH2 of the alkyl chain), 0.8 (t, CH3 of the alkyl chain). Polymer Analysis. The number-average molecular weight M̅ n and number-average degree of polymerization DPn of protected NAhPols were determined by UV absorbance.15,16 The three benzoyl groups of the transfer reagent (TR) strongly absorb at λ = 272 nm (molar absorptivity coefficient ε272 = 2.8 × 103 L.mol−1.cm−1). A calibration 4627
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dM were determined using a Zetasizer Nano-S model 1600 (Malvern Instruments Ltd., UK) equipped with a He−Ne laser (λ = 633 nm, 4.0 mW). The time-dependent correlation function of the scattered light intensity was measured at a scattering angle of 173° relative to the laser source. NAhPol stock solutions were prepared at 100 g·L−1 in Milli-Q water and were stored overnight at room temperature. On the day of the experiment the solutions were filtered through a 0.45 μm PVDF syringe filter, diluted to the final concentration, and the size of the particles was measured 1 h after filtration. Other conditions were as reported elsewhere.21 Aqueous Size-Exclusion Chromatography (ASEC). NAhPol stock solutions were prepared at 100 g·L−1 in Milli-Q water under magnetic stirring overnight at 4 °C and then diluted 10 times in 100 mM NaCl 20 mM Tris buffer (pH 8). A 100 μL of the latter solution was injected onto a Superose 12 10−300GL column (bed volume: 20 mL; void volume: 7.5 mL; separation range: DH = 2− 18 nm) connected to a Ä kta purifier 10 system (GE-Healthcare) and calibrated according to the procedure reported elsewhere.22 The column was equilibrated with (a) 20 mM Tris/HCl buffer, 100 mM NaCl, pH 8, for analyzing NAhPols particles and OmpX/A8−35 complexes; (b) 20 mM phosphate buffer, 100 mM NaCl, pH 7, for BR/ NAhPols complexes; and (c) 20 mM phosphate buffer, 100 mM NaCl, pH 6.75, for OmpX/NAhPols complexes. Experiments were run at room temperature. The flow rate was set at 0.5 mL·min−1 and elution profiles followed at 220, 280, and/or 554 nm. Buffers in Density, AUC, and SANS Measurements. Buffer H: 100 mM NaCl, 20 mM NaH2PO4/Na2HPO4, pH 7.4 in Milli-Q water. Buffer D: 100 mM NaCl, 20 mM NaH2PO4/Na2HPO4 in >99% 2 H atom sterile filtered D2O (Spectra Gases, Inc.). The pH was 7.4 for the analysis with NA29 and 6.8 for the analysis with NA11. Buffer H and Buffer D for NA11 contained in addition 0.02% NaN3. NAhPols Sample Preparation for Density, AUC, and SANS Measurements. NAhPols were placed at 4 °C in the dark and under vacuum in a desiccator and were dried over phosphorus pentoxide for two months. Solutions at 5, 10, and 15 g·L−1 were prepared in H2O or D2O by precise weighing of the powder, solvents, and solutions. Complementary experiments were done, in Buffer H and Buffer D, with NA29 at 5 g·L−1 and NA11 at 5−15 g·L−1. Determination of the Partial Specific Volume from Density Measurements. Those were performed using a density-meter DMA5000 (Anton PAAR, Graz Austria). Partial specific volume, v,̅ was obtained from solvent and solution densities, ρ° and ρ (g.mL−1), measured at concentration c (g.mL−1) according to
(ρ − ρ°) = (1 − ρ° v ̅ )/c
In deuterated solvent, Svedberg equation is slightly modified in view of the increase of mass of the particlefrom M to MDrelated to H-D exchange
s = M(MD/M − ρv ̅ )/(NA 3πηD H)
These two equations were used to calculate s20w from the experimental values of s. The frictional ratio f/f min relates DH and the diameter of the anhydrous particle volume Dmin
DH = f /fmin Dmin
(7)
Fringes displacement J characterizing the boundary is related to the refractive index increments ∂n/∂c (mL/g) and species concentration c (g.mL−1), with λ the wavelength of the laser (675 × 10−7 cm in our experiments), according to
(∂n/∂c) = (J /c)(λ /l)
(8)
For the study of the complexes, the protein concentration was derived from the absorbance signal at 280 nm, and the amount of bound NAhPol from the combination of fringe shift and absorbance signals. The derived composition was used for the calculation of the partial specific volume of the complex for the calculation of s20w and f/f min. As numerical values, for the c(s) analysis, we typically used 200 generated sets of data on a grid of 300 radial points, calculated using frictional ratio of typically 1.2 sometimes fitted, and a regularization procedure with a confidence level of 0.68, and considered v ̅ determined by density and v ̅ = 0.76 mL.g−1 for the samples of NAhPol and complexes, respectively. We measured for H2O, D2O, and hydrogenated and deuterated buffer densities of 0.9982, 1.1053, 1.0052, and 1.1116 g.mL−1 and viscosities of 1.002, 1.295, 1.079, and 1.299 mPa.s, using a density-meter DMA 5000 and viscosity-meter AMVn (Anton PAAR, Graz Austria). MD/M = 1.015 was estimated for NA29 in 100% D2O, considering 10 exchangeable H per monomer in addition to 4 others for the whole polymer, thus 424 exchangeable H for a macromolecule of 28 899 Da. Numerical values for BR are given in ref 24. Numerical values used for dOmpX are as follows: molar mass of the monomer in Buffer H: 17 161 g·mol−1; MD/M = 1.012 corresponding to 80% of exchanged exchangeable hydrogens; v ̅ = 0.68 mL.g−1; (∂n/∂c) = 0.187 mL.g−1; molar extinction coefficient at 280 nm: 31 860 M−1·cm−1. SANS Experiments and Raw Data Reduction. NAhPols samples and the respective buffers (all ∼160 μL) were prepared at several H2O/D2O ratios (0%, 30%, 70%, and 100% D2O for NA11 at 15 g·L−1; 0%, 10%, 20%, 30%, 60%, 70%, 80%, and 100% D2O for NA29 at 15 g·L−1, and at 5 and 10 g·L−1 at 0% and 100% D2O, respectively). The BR/NA29 complexes were measured at three different protein concentrations: 8.85, 4.42, and 2.20 g·L−1 (in Buffer H), and 9.70, 4.80, and 2.40 g·L−1 (in Buffer D). Measurements were done at 20 °C on the instrument D22 at the Institute Laue-Langevin (ILL) (Grenoble, France) in Hellma quartz cuvettes 100QS with 1 mm optical path length. Scattering data were recorded with a wavelength of λ = 6 Å at an instrumental collimator/detector configuration of 2 m/2 m (NA29 and complexes) and of 1.4 m/1.4 m and 5.6 m/5.6 m (NA11). At each configuration, the H2O/D2Obuffers, the empty beam, an empty quartz cuvette, and a boron sample (electronic background) were measured. Exposure times varied from 30 min to 2 h for individual samples, transmission measurements about 3 min. The raw data were reduced with a standard ILL software package.25 The corrected scattered intensities I(Q) (with the scattering vector Q = (4π/λ)sinθ, where 2θ is the scattering angle) from different Q-ranges were merged and the buffer signals subtracted using the program “PRIMUS”.26 SANS Data Analysis. The radii of gyration, Rg, and the intensities in the forward scattering direction, I(0), of all samples were extracted by the Guinier approximation, in the valid range of RgQ ≤ 1.327
(4)
Analytical Ultracentrifugation. Sedimentation velocity experiments were performed using a Beckman XL-I analytical ultracentrifuge and an AN-50 TI rotor (Beckman Coulter, Palo Alto CA USA). The experiments were carried out at 20 °C and at 42 000 rpm, with cells with double sector centerpiece of 1.2 or 0.3 cm optical path (l), filled typically with ∼420 or 100 μL of sample and of reference solvent. Scans were recorded every ∼5 or 10 min, overnight, using interference optics, and, for the protein samples, at 280 nm, and for BR analysis 555 nm. The sedimentation profiles were analyzed by the sizedistribution analysis of Sedfit (free available at http://www. analyticalultracentrifugation.com). In Sedfit, finite element solutions of the Lamm equation for a large number of discrete, independent species, for which a relationship between mass, sedimentation, and diffusion coefficients, s and D, is assumed, are combined with a maximum entropy regularisation to represent a continuous sizedistribution.23 The linear fit s/s0 = (1 − ksc) provided an estimate of the sedimentation, s0, at infinite dilution, and the concentration dependency factor, ks. The s-value depends in the ideal case on the mass, M, and hydrodynamic diameter, DH, of the particle, and on the solvent density, ρ, and viscosity, η, according to the Svedberg equation
s = M(1 − ρv ̅ )/(NA 3πηDH)
(6)
ln[I(Q )] = ln[I(0)] − 1/3R g 2Q 2
(5) 4628
(9)
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dihexanoylphosphatidylcholine (DHPC, Avanti Polar Lipids) at 4 °C. The final protein concentration was 0.46 g·L−1 in 2% (w/v) DHPC. Preparation of Amphipol/Membrane Protein Complexes. Bacteriorhodopsin. Final proteins preparations were incubated 15−20 min at 4 °C with aliquots of NAhPols or A8−35 stock solutions (100 g·L−1 in water) to reach the final protein/ polymer mass ratio (1:5 or 1:10 w/w for ASEC analysis, 1:5 w/w for AUC and SANS experiments). The detergent was removed by incubation with polystyrene beads (Bio-Beads SM2, 10 g per g detergent) for 2 h under gentle stirring on a wheel. After elimination of the beads and a 20 min centrifugation at 100 000 × g in the TLA 100.2 rotor of a TL 100 ultracentrifuge (Beckman Coulter France), the protein concentrations were determined spectroscopically. For AUC and SANS measurements on BR/NA29 complexes, a 40 mL sample at 0.25 g·L−1 BR was concentrated using 4 mL centrifugal filter Amicon Ultra 30 000 MWCO (Millipore) and by running several centrifugations in Buffer H to a final volume of 1 mL at ∼9 g·L−1 BR. 500 μL of this final sample was used for SANS and AUC experiments. The remaining 500 μL was diluted eight times in Buffer D and centrifuged (5 min at 3000 × g) in Amicon Ultra filter tubes (Beckman rotor JA-25.50). The procedure was repeated twice. The final sample (500 μL of BR/NA29 at ∼10 g·L−1 in Buffer D) was used for SANS and AUC experiments. AUC experiments were performed with diluted samples (5 and 15 μL of each sample diluted to 120 μL in the appropriate buffer). SANS experiments were performed without dilution and with samples diluted 2/3 and 1/3 from the stock solutions. The samples at 30% D2O was prepared from Buffer D and the stock samples in Buffer H. OmpX. OmpX trapping by NAPols was performed as reported for trapping the transmembrane domain of outer membrane protein A37 with A8−35, i.e., at a 1:4 OmpX/NAPol ratio (w/w). DHPC was removed with BioBeads: after 30 min incubation in the presence of NAPols, the beads were added at a 10:1 bead/detergent mass ratio. Beads were removed by centrifugation after 3 h of incubation at room temperature. In order to remove any residual traces of DHPC, 10 cycles of dilution/concentration were performed using a centrifugal filter unit (10 kDa cutoff, Amicon, Millipore) with the NMR buffer: 20 mM phosphate buffer, 100 mM NaCl, 0.05% NaN3 (pH 6.8) in 10% D2O. The final sample contained ∼1.3 mM dOmpX.
The masses of NAhPols particles, M, were determined from the I(0) intensities in 100% H2O and 100% D2O according to28
M=
1 − TW I(0) NA103 [b − ρ N 0v ̅ ]−2 f 4πTS Iinc(0) Ct
(10)
Iinc(0) and TW = 0.523 are the incoherent scattering in the forward direction and the neutron transmission rate of 1 mm optical path water, respectively, TS is the neutron transmission rate of the sample, C the NAhPol concentration in mg.mL−1, t = 1 mm is the thickness of the quartz cuvette, f = 0.81 at 6 Å is a correction factor for the anisotropicity of the solvent scattering, ρN0 is the solvent neutron scattering length density in cm−2, and b is the scattering length density of the polymer, in cm/g. We used ρN0 = −5.62 × 109/6.40 × 1010 cm−2 for H2O and Buffer H/D2O and Buffer D; b = 8.84 × 109/1.79 × 1010 cm/g in H2O/D2O, for NAhPol from the chemical composition of NA29 (see above) and considering a full exchange. For NA29, I(0) at infinite dilution was extrapolated from the linear regression of C/I(0) versus C.29 The contrast match points were determined experimentally by the intersection of a linear fit through all points with the abscissa of (I(0)/[TSC])1/2 as a function of D2O content. The pair distance distribution function, p(r), was determined for NA29 at 15 g·L−1 in 100% H2O and 100% D2O using the program GNOM in user default mode.30 For an ideal particle of volume V, p(r) = 4πr2Vγ(r), with γ(r) the autocorrelation function defined as the average of the product of the two scattering density contrasts (cm−2) separated by a distance r. The final 1D SANS curves corresponding to the BR/NA29 complexes alone (without contribution from free micelles) were obtained in two steps: (1) extrapolation of the scattering curves in both Buffer H and Buffer D at infinite dilution using the concentration series and the protocol developed by Costenaro et al.29 (2) Subtraction of the free NAhPol contribution from their extrapolated curves in H2O and D2O, respectively. Analysis of the forward intensities was made according to ref 24. The p(r) from the BR monomer (PDB entry 1QHJ) was obtained with GNOM from its back-calculated scattering curve using the program CRYSOL.31 Those corresponding to the BR/NA29 complexes were extracted with GNOM from the reduced scattering curves (as described above). The low-resolution model of the BR/NA29 complex was obtained using the program DAMMIF32 from the complex data in Buffer D. DAMMIF uses a single phase with homogeneous contrast to the solvent to model a low-resolution shape. We chose Buffer D rather than Buffer H since in the former the contrast of all components (BR, NAPol, and lipids) was highest and most homogeneous. The shape therefore represents an approximation of a slightly heterogeneous system. It reflects the global shape of the complex and the structural details must not be overinterpreted. The high-resolution monomer was superposed by hand into the overall density. Protein Purification. Bacteriorhodopsin. H. salinarum cells (S9 strain, a gift of G. Zaccai,̈ IBS Grenoble) were grown under light at 37 °C in a liquid growth medium containing 12 g·L−1 ̈ peptone (LP0037, Oxoid) as described in ref 33. Purple membrane (PM) was isolated as described in ref 34 and stored at −80 °C. Further steps were performed at 4 °C in the dark. PM containing BR at 6 g·L−1, suspended in 20 mM sodium phosphate buffer, pH 7.0, was solubilized by incubation during 40 h at 4 °C in the dark with 100 mM octylthioglucoside (OTG; cmc ∼9 mM). After centrifugation for 20 min at 200 000 × g in the TLA 100.2 rotor of a TL 100 ultracentrifuge (Beckman Coulter France), the supernatant was diluted to 18 mM OTG with detergent-free buffer. Aliquots of NAhPols stock solutions (100 g·L−1 in water) were added to 250 μL samples to reach final protein/polymer mass ratios of 1:5 or 1:10. OmpX. Overexpression of uniformly 2H,13C, and 15N-labeled OmpX (hereafter, dOmpX, labeled in view of its use for NMR experiments) was prepared as described in ref 7. Briefly, the protein was expressed in E. coli and purified from inclusion bodies using procedures similar to those described in refs 35 and 36. Inclusion bodies were solubilized in 6 M urea, 20 mM Tris-HCl, 5 mM EDTA, pH 8.5. OmpX was refolded by slow dilution into a solution of
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RESULTS Synthesis of NAhPols. The synthesis of NAhPols is based on three key steps: (i) synthesis of the thiol-based transfer reagent (TR),15 (ii) synthesis of the diglucosylated amphiphilic protected monomer (pM),20 and (iii) free-radical homotelomerization (Scheme 2). The synthesis of the transfer reagent (TR) was performed as previously described,15 starting from 3-mercaptopropionic acid. Briefly, the thiol function was protected by a trityl group, followed by condensation of tris-(hydroxymethyl)-amidomethane (Tris) onto the carboxylic acid group. The three hydroxyl groups of Tris were next protected by benzoyl groups and the trityl protective group was finally removed to lead, after purification, to the transfer reagent in ∼40% overall yield, in four steps. Thanks to the aromatic groups grafted onto Tris, determination of the molecular mass of the final polymers can be achieved by UV and 1H NMR measurements. The key molecule of NAhPol preparation is the di-O-acetylated glucose-based THAM monomer pM endowed with an undecyl chain, which was synthesized as recently reported.20 First, two hydroxyl groups of THAM were protected by reaction with dimethoxypropane. The remaining hydroxyl group was then hydrophobized by reaction with undecylisocyanate in basic conditions. Subsequent removal of the 1,3-dioxane protective group followed by diglucosylation 4629
dx.doi.org/10.1021/la205026r | Langmuir 2012, 28, 4625−4639
Langmuir
Article
in the less polar mixed-solvent system. Such an effect may be due to hydrophobic associations of polymer chains and the formation of microdomains, which in turn could lead to a micellar catalysis of the reaction.39 Considering the lower polarity of THF (ε = 7.6) compared to that of acetonitrile (ε = 37.5), the formation of hydrophobic domain during the polymerization of the protected monomer pM in refluxing THF is therefore more likely. This may lead to higher rate of polymerization allowing a complete consumption of pM within 18 h of reaction despite its bulkiness. On the contrary, in the very polar acetonitrile the micellar catalysis is not favored leading to very low rate of polymerization. The batch-to-batch reproducibility in THF was good, with similar overall yields and a comparable molecular weight of the resulting polymers. Acetylated NAhPols were purified by preparative size exclusion chromatography (SEC) in organic solvents on hydroxylpropylated cross-linked dextran. The purified acetylated polymers were readily soluble in usual organic solvents such as methanol, chloroform, and toluene, as well as in DMF or DMSO. Simultaneous deprotection of the acetyl and benzoyl groups was achieved under the Zemplén conditions, yielding the amphiphilic polymers (Scheme 2). Pure NAhPols were obtained in ∼50% yield after purification by dialysis against water using cellulose membranes, followed by lyophilization. Throughout the present article, NAhPols are denoted by a short name reflecting their chemical structure: the letters NA stand for ″nonionic amphiphilic homopolymer″, followed by the average molecular weight expressed in kDa. Batch numbers are given in Table 1. Structure Determination. The M̅ n of the polymers was determined by two methods: (i) by determining the ratio of the mass concentration of acetylated NAhPol solutions to the molar concentration, determined from the UV absorption (λmax = 272 nm), of the three benzoyl groups grafted at the end of the polymer backbone (Scheme 2); (ii) by comparing the integrated values of the 1H NMR signal of the benzoyl groups to those of the NH vicinal methylene of each amphiphilic monomer constituting the polymer backbone (Figure 1). A good correlation was observed between the two techniques (Table 1), particularly for the shorter polymers. For the protected NA11, for instance, M̅ n values determined by the UV and NMR techniques were 17 and 18 kDa, respectively. For longer polymers, the difference observed between the two spectroscopic techniques was slightly larger but remained
