Lactobionamide Surfactants with Hydrogenated, Perfluorinated or

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Langmuir 2006, 22, 8881-8890

8881

Lactobionamide Surfactants with Hydrogenated, Perfluorinated or Hemifluorinated Tails: Physical-Chemical and Biochemical Characterization Florence Lebaupain,†,| Andre´s G. Salvay,‡,|,⊥ Blandine Olivier,§,| Gre´gory Durand,§ Anne-Sylvie Fabiano,§ Nicolas Michel,§ Jean-Luc Popot,† Christine Ebel,*,‡ Ce´cile Breyton,*,† and Bernard Pucci*,§ C.N.R.S., UniVersite´ Paris-7, UMR 7099 Institut de Biologie Physico-Chimique, 13 rue Pierre-et-Marie-Curie F-75005 Paris France, Institut de Biologie Structurale, UMR 5075 CEA-CNRS-UniVersite´ Joseph Fourier, 41 rue Jules Horowitz, F-38027 Grenoble Cedex 1, France, and Laboratoire de Chimie Bioorganique et des Syste` mes Mole´ culaires Vectoriels, UniVersite´ d’AVignon et des Pays du Vaucluse, Faculte´ des Sciences, 33 rue Louis Pasteur, F-84000 AVignon, France ReceiVed April 21, 2006. In Final Form: July 18, 2006 Detergents are customarily used to solubilize cell membranes and keep membrane proteins soluble in aqueous buffers, but they often lead to irreversible protein inactivation. Hemifluorinated amphiphiles with hybrid hydrophobic chains have been specifically designed to minimize the denaturating propensity of surfactants toward membrane proteins. We have studied the physical-chemical and biochemical properties of lactobionamide surfactants bearing either a hydrogenated, a fluorinated or a hemifluorinated chain (respectively H-, F-, and HF-Lac). We show that the dual composition of the hydrophobic chain of HF-Lac endows it with unusual physical-chemical properties as regards its critical micellar concentration, interfacial area per molecule, and behavior upon reverse phase chromatography. Analytical ultracentrifugation shows that, whereas H-Lac assembles into well-defined micelles, F-Lac and HF-Lac form large and heterogeneous assemblies, whose size increases with surfactant concentration. Molecular dynamics calculations suggest that F-Lac forms cylindrical micelles. The ability of HF-Lac to keep membrane proteins soluble was examined using the cytochrome b6 f complex from Chlamydomonas reinhardtii's chloroplast as a model protein. HF-Lac/b6 f complexes form particles relatively homogeneous in size, in which the b6 f complex is as stable or markedly more stable, depending on the surfactant concentration, than it is in equivalent concentrations of hydrogenated surfactants, including H-Lac.

Introduction Biological membranes are composed of lipids, which autoassociate to form a bilayer, and membrane proteins, which interact with lipids through their hydrophobic transmembrane domain. This results in a thermodynamically stable supramolecular assembly. Membrane proteins, which may comprise either one or several polypeptide chains (subunits) and hydrophobic cofactors, need to be extracted from the membrane for most in vitro studies. This is commonly done thanks to detergents, which bind, in place of the lipids, to the transmembrane surface of the proteins, making them water-soluble. However, the dissociating effect of detergents can be difficult to control, resulting in the destabilization and irreversible inactivation of the solubilized protein. Two likely mechanisms leading to inactivation are the intrusion of the detergent into the transmembrane region of the protein and/or the dissociation of stabilizing lipids, cofactors, or subunits.1-5 In an attempt at overcoming these problems, we are * Corresponding authors. E-mail: [email protected]; cecile.breyton@ ibpc.fr; [email protected]. † UMR 7099 Institut de Biologie Physico-Chimique. ‡ UMR 5075 Institut de Biologie Structurale. § Universite ´ d’Avignon et des Pays du Vaucluse. | These authors have contributed equally to the work. ⊥ Present address: Instituto de Fı´sica de Lı´quidos y Sistemas Biolo ´ gicos, Universidad Nacional de La Plata, c.c. 565, B1900BTE La Plata, Argentina. (1) le Maire, M.; Champeil, P.; Møller, J. V. Biochim. Biophys. Acta 2000, 1508, 86-111. (2) Bowie, J. U. Curr. Opin. Struct. Biol. 2001, 11, 397-402. (3) Garavito, R. M.; Ferguson-Miller, S. J. Biol. Chem. 2001 276, 3240332406. (4) Seddon, A. M.; Curnow, P.; Booth, P. J. Biochim. Biophys. Acta 2004, 1666, 105-117.

studying the potential of fluorocarbon surfactants.6 Our rationale is the following: alkanes and perfluorinated alkanes, although they are both hydrophobic, are poorly miscible; for this reason, surfactants with fluorinated alkyl chains are lyophobic: they do not partition well into biological membranes, and therefore, have little cytolytic effect.7,8 For the same reason, they are poor solvents for lipids and hydrophobic cofactors and can thus be expected to be less delipidating. With their hydrophobic moieties being lyophobic, more bulky, and more rigid than their hydrogenated counterparts, they may also be expected to intrude less easily into the protein structure itself. In a previous work, we synthesized various amphiphilic fluorocarbon monoadducts or telomers derived from trishydroxymethyl aminomethane. Membrane proteins, initially extracted using hydrogenated detergents, remained soluble after being transferred into one of them (F8-telo-H in ref 6; hereafter F-TAC), provided the surfactant was used at rather high concentration. However, the proteins aggregated with time, suggesting that surfactant/protein interactions did not compete efficiently with protein/protein ones.6 A hydrocarbon tip, an ethyl group, was therefore added to the fluorocarbon tail of F-TAC, in an attempt at improving interactions between the protein transmembrane surface and the surfactant.9 The hemifluorinated (5) Breyton, C.; Tribet, C.; Olive, J.; Dubacq, J.-P.; Popot, J.-L. J. Biol. Chem. 1997, 272, 21892-21900. (6) Chabaud, E.; Barthe´le´my, P.; Mora, N.; Popot, J.-L.; Pucci, B. Biochimie 1998, 80, 515-530. (7) Barthe´le´my, P.; Tomao, V.; Selb, J.; Chaudier, Y.; Pucci, B. Langmuir 2002, 18, 2557-2563 and references cited. (8) Pucci, B.; Maurizis, J.-C.; Pavia, A. A. Eur. Polym. J. 1991, 27, 11011106.

10.1021/la061083l CCC: $33.50 © 2006 American Chemical Society Published on Web 09/12/2006

8882 Langmuir, Vol. 22, No. 21, 2006

surfactant thus obtained, HF-TAC, was found to keep in solution, under their native form, a variety of membrane proteins of different types, function, and origin.10 Besides their mildness, which ought to make them suitable to studying fragile membrane proteins or membrane protein complexes, hemifluorinated surfactants open the way to original experiments, impossible to carry out in detergent solution, such as presenting a solubilized protein to a preformed membrane without solubilizing the latter.11 Because the synthesis of the polar head of HF-TAC involves radical polymerization, it is inevitably polydisperse, which can lead to batch-to-batch variations. To circumvent this problem, we have undertaken to synthesize hemifluorinated surfactants with chemically defined polar heads. Surfactants bearing an aminoxide polar head confirmed the interest of the approach.12 They proved less mild than HF-TAC, however, presumably because of the small size of the polar head. We thus undertook to synthesize surfactants comprising a disaccharide moiety, which is highly soluble, bulky, neutral, and constitutes the polar head of several detergents commonly used in biochemistry, such as n-dodecyl-β-D-maltopyranoside (dodecylmaltoside, hereafter DDM). We report here on the synthesis and a comparative physicalchemical and biochemical study of hydrocarbon, fluorocarbon, or hydrocarbon end-capped fluorocarbon lactobionamide surfactants (respectively denoted H-, F-, and HF-Lac). As a test membrane protein, we used cytochrome b6 f, one of the major complexes of photosynthetic oxygenic chains. When purified from thylakoid membranes of Chlamydomonas reinhardtii, a freshwater alga, functional b6 f complexes are present as dimers, each monomer comprising eight subunits, seven cofactors, and several lipids.13 The b6 f complex represents a good test system for determining whether novel surfactants are mild or not, because it is particularly fragile and easily dissociates into inactive monomers when handled in detergent solution.5 Materials and Methods Chemistry. General. Progress of the reactions and homogeneity of the compounds were monitored by thin-layer chromatography (TLC; Merck 254 silica plates). Compounds were detected either by exposure to ultraviolet light (254 nm) or by spraying with a 5% sulfuric acid solution in methanol and/or 5% ninhydrin solution in ethanol (detection of amine-containing compounds) and heating at 150 °C. Purification was performed by flash column chromatography over silica gel (Merck 60). High-pressure liquid chromatography (HPLC) purifications were performed on a Varian Microsorb C18 column (5 µm granulometry, 21.4 × 250 mm) with acetonitrile/ water (35/65 v/v) at a flow rate of 16 mL‚min-1, with detection at 215 nm. All solvents were removed under vacuum. 1H, 13C, and 19F NMR spectra were recorded on a Brucker AC 250 spectrometer. Chemical shifts are given in ppm relative to tetramethylsilane, using the deuterium signal of the solvent (CDCl3) as a heteronuclear reference for 1H and 13C. Mass spectra were recorded on a DX 300 JEOL instrument. All commercial solvents were distilled and dried according to standard procedures. Syntheses. Lactobionolactone was prepared by dehydration of commercial lactobionic acid.14 Surfactants bearing a lacto(9) Barthe´le´my, P.; Ameduri, B.; Chabaud, E.; Popot, J.-L.; Pucci, B. Org. Lett. 1999, 1, 1689-1692. (10) Breyton, C.; Chabaud, E.; Chaudier, Y.; Pucci B.; Popot J.-L. FEBS Lett. 2004, 564, 312-318. (11) Palchevskyy, S. S.; Posokhov, Y. O.; Olivier, B.; Popot, J.-L.; Pucci, B.; Ladokhin, A. S. Biochemistry 2006, 45, 2629-2635. (12) Chaudier, Y.; Zito, F.; Barthe´le´my, P.; Stroebel, D.; Ameduri, B.; Popot, J.-L.; Pucci, B. Bioorg. Med. Chem. Lett. 2002, 12, 1587-1590. (13) Stroebel, D.; Choquet, Y.; Popot, J.-L.; Picot, D. Nature (London) 2003, 426, 413-418. (14) Williams T. J.; Plessas, N. R.; Goldstein, I. J. Arch. Biochem. Biophys. 1979, 195, 145-151.

Lebaupain et al. Scheme 1. Synthetic Pathways of Lactobionamide Derived Surfactantsa

a (a) NaN (1 eq), DMF, 24 h (39%); the residual di-odo compound 3 is re-cycled. (b) H2, Pd/C, MeOH, 48 h (98%). (c) Lactobionolactone, TEA (pH ) 9)/methoxyethanol, 65 °C, 24 h (51%).

bionamide group as polar head and a hydrophobic tail derived from either dodecyl-, decyl-, 1H,1H,2H,2H-perfluorooctyl-, or 3F,3F,4F,4F,5F,5F,6F,6F,7F,7F,8F,8F-decylamine were synthesized, yielding, respectively, H12-Lac, H10-Lac, F-Lac, and HF-Lac (Scheme 1). The synthesis of H12-Lac, H10-Lac, and F-Lac has already been described.14-16 It follows the synthetic scheme proposed by Williams et al,14 as summarized in Scheme 1. Surfactants were purified by HPLC before any physical-chemical characterization or biochemical application. HF-Lac was prepared following the same procedure. 1,10-Diiodo-3,3,4,4,5,5,6,6,7,7,8,8-decafluorodecane, kindly provided by Dr B. Ameduri from ENSCM-Montpellier, France, was used as starting material.17 The first step is the selective substitution of one of the two iodine atoms (Scheme 1). This reaction is carried out by using one equivalent of sodium azide in dimethylformamide (DMF), leading to the azido derivative with a yield of 39%. The material that has not reacted is recovered by chromatography on silica gel and resubmitted to nucleophilic substitution. Hydrogenation in methanol in the presence of catalytic amounts of Pd/C leads to the simultaneous reduction of the iodo and azido groups, yielding the hemifluorooctylamine. This compound is immediately condensed on the lactobionolactone in boiling methoxyethanol at pH 9. After purification by HPLC, HF-Lac is isolated as a white solid with an overall yield of 20%. Compounds were structurally characterized by their 1H, 13C, and 19F NMR spectra. Chemical shifts (δ) for H- and F-Lac were in good agreement with earlier analyses.14-16 Those for HF-Lac are as follows: NMR 1H (DMSO-d6): δ (ppm) 7.96 (t, 1H, NH, 3JH-H ) (15) Emmanouil, V.; El Ghoul, M.; Andre-Barres, C.; Guidetti, B.; RicoLattes, I.; Lattes, A. Langmuir 1998, 14, 5389-5395. (16) El Ghoul, M.; Escoula, B.; Rico I.; Lattes A. J. Fluor. Chem. 1992, 59, 107-112. (17) Manseri, A.; Ameduri, B.; Boutevin, B.; Kotora, M.; Hajeck, M.; Caporriccio, G. J. Fluor. Chem. 1995, 73, 151-158.

Characterization of Lactobionamide Surfactants 6.4 Hz); 5.23-3.31 (m, 15H, CH2NH + H sugar); 2.33 (m, 4H, CH2(CF2)6CH2CH2); 1.07 (t, 3H, CH3, 3JH-H ) 7,7 Hz); NMR 19F (DMSOd6): δ (ppm) -114.2 (CF2CH2CH2NH); -116.7 (CF2CH2CH3); -122.2 (CF2CF2(CF2)2CF2CF2); -124.0 (CF2CF2(CF2)2CF2CF2); NMR 13C (DMSO-d6): δ (ppm) 173.2 (CO); 118.5-110.7 ((CF2)6); 105.0 (C1); 83.1 (C4); 76.2 (C2); 73.7 (C3′); 72.5 (C5′); 71.8 (C5); 71.6 (C3); 70.9 (C2′); 68.7 (C4′); 62.8 (C6); 61.7 (C6′); 42.0 (CH2NH); 30.6 (CH2CH2NH); 24.2 (CH3CH2); 4.8 (CH3). HRMS FAB+[M+Na+]: 736.1531; calculated 736.1603. Biochemistry. Cytochrome b6 f was purified in 0.2 mM DDM according to ref 13. Briefly, the solubilization supernatant of thylakoid membranes was loaded onto a Source Q column and eluted with a salt gradient. The fractions containing the b6 f were further purified on a Nickel column, and the b6 f was finally desalted by chromatography. Final preparations contain 0.2 mM free DDM and ∼260 molecules of bound DDM per b6 f dimer,5 i.e., typically, ∼0.55 mM of DDM altogether for a 2.6 µM b6 f sample. For analytical ultracentrifugation (AUC) experiments, purified b6 f was supplemented with 150 mM NaCl and either HF-Lac (2 or 5.5 mM) or DDM (5 mM). In HF-Lac samples, DDM was removed by incubation with polystyrene beads (BioBeads SM2) during 2 h at 4 °C. TLC analysis showed the presence of residual amounts of DDM (∼0.2 mM) after incubation with BioBeads at either HF-Lac concentration (DDM removal on the beads was not affected by the presence of HF-Lac). Preparative centrifugation was carried out for 4 h at 200 000 × g in 2 mL of 10-30% (w/w) sucrose gradients containing 20 mM Tris buffer, pH 8.0, and either DDM or HF-Lac at the desired concentration.10 In the latter case, b6 f samples were supplemented with 1 mM HF-Lac and incubated 15-30 min prior to being loaded onto the gradients. The colored bands, containing the protein, were collected with a syringe and kept on ice in the dark for activity measurements.18 Tensiometry. Surface tension measurements were carried out at 25 °C with a KRUSS K12 tensiometer using a Wilhelmy plate. The surface excess Γ at the air-water interface was calculated by the Gibbs adsorption isotherm equation Γ ) -(1/RT)(∂γ/∂lnC), where γ is the surface tension (N‚m-1) at the surfactant concentration C (mol‚m-3). The occupied area (A) per surfactant molecule is calculated from A ) 1/NAΓ, where NA is Avogadro’s number. Fluorescence. Critical micellar concentration (CMC) of H12-Lac was determined from steady-state fluorescence measurements.19 Measurements were carried out at 25 ( 0.1 °C on a SPEX-Fluoromax 2 fluorimeter (Jobin-Yvon). Fluorescence emission spectra of samples containing 1.6 µM pyrene were recorded using an excitation wavelength of 335 nm. Emission intensities were recorded at I1 ) 373 nm and I3 ) 384 nm.19 Determination of log k′W Values. Compounds were dissolved in acetonitrile/water (5/5 v/v) at 2.0 g‚L-1 and injected onto a Microsorb C18 column (5 µm granulometry, 250 × 4.6 mm) at 27 °C. Three runs were performed with different methanol/water ratios (9/1 to 7/3 (v/v)) at a flow rate of 0.8 mL‚min-1. Compounds were detected at 215 nm. log k′ was calculated as log k′ ) log((t - t0)/t0), where t is the retention time of the surfactant and t0 the elution time of methanol, which is not retained on the column. log k′W values were deduced by extrapolation of the linear regression (r2 > 0.997) to 100% water.20,21 Dynamic Light Scattering. Particles size and polydispersity were measured at 25 °C using a Zetasizer Nano-S model 1600 (Malvern Instruments Ltd., U. K. ) 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 (backscattering detection). The Stokes radius (RS) of the particles was estimated from their diffusion coefficient (D) using the Stokes-Einstein equation D ) kBT/6πηRS, where kB is Boltzmann’s constant, T the absolute temperature, and η the viscosity of the solvent. Stock solutions of HF-Lac at 20 mM were prepared (18) Pierre, Y.; Breyton, C.; Kramer, D.; Popot J.-L. J. Biol. Chem. 1995, 270, 29342-29349. (19) Arai, T.; Takasugi, K.; Esumi, K. Colloids Surf., A 1996, 119, 81-85. (20) Braumann, T. J. Chromatogr. 1986, 373, 191-225. (21) Hseih, M.-M.; Dorsey, J. G. Anal. Chem. 1995, 67, 48-57.

Langmuir, Vol. 22, No. 21, 2006 8883 at 25 °C by dissolving under hand-shaking. No ultrasonication was applied. The size of the particles was measured both 1 h after preparation of the solutions and 1 week later. Preparation of Samples for Density and Sedimentation Measurements. Surfactants were dried over P2O5 during 3 days. The solutions were then prepared by dissolution of the weighted solid surfactant in H2O. Density Measurements. The solution and solvent densities, F and F° (g‚mL-1) were measured at 20 °C with a DMA5000 density meter (Anton Paar, Graz, Austria, accuracy 2.10-6 g‚mL-1). The apparent partial specific volume νj (mL‚g-1) was obtained from the linear dependency of F - F° over the concentration of surfactant, (F - F°)/c ) (1 - F°νj), using, typically, five concentrations (c) in the range of 10-3-10-2 g‚mL-1. We proposed a new method, based on the analysis of sedimentation velocity experiments performed in H2O and D2O solvents.22,23 The hypothesis is that the size distribution (i.e., aggregation number and dimension) in the sample is the same in H2O and D2O. In view of the complex behavior of HF-Lac and F-Lac, it is not sure the assumption would be valid, and the approach was not used. Analytical Ultracentrifugation. Sedimentation velocity experiments were performed in a Beckman XLI ultracentrifuge at a rotor speed of 42 000 rpm. The sample and reference solvent (400 µL each) were placed in a double-sector cell of optical path 1.2 cm equipped with sapphire windows. The concentrated samples of HFLac (25.6 and 38.4 mM) were loaded into a cell with a 0.3 cm optical path. Measurements were carried out for more than 15 h, at 20 °C for the surfactants and at 4 °C for the b6 f/surfactant complexes. Sedimentation profiles were acquired every 2 min using interference optics (all samples) and 280- and 420-nm absorption spectra (b6 f/ surfactant complexes). Analysis of the data was made according to the c(s) analysis method, using the program Sedfit24 (version 8.9, available at www.analyticalultracentrifugation.com). The analysis took into account 60 experimental profiles over a total of 15 h of sedimentation, by simulating the Lamm equation for 300 particles in the 0.01-20 S range for the surfactant and in the 1-70 S range for the b6 f/surfactant complexes. The c(s) method deconvolutes the effects of diffusion broadening, yielding high-resolution sedimentation coefficient distributions. This is done by assuming a plausible relationship between sedimentation and diffusion coefficients, s and D. It has to be mentioned that an inadequate relationship between s and D only decreases the details and resolution of the distribution but does not affect the values of s and, therefore, does not bias subsequent estimates of surfactant binding. The s-D relation is based on the values of the particle partial specific volume, νj, frictional ratio, f/f°, solvent density, F°, and viscosity, η. The details are given in the Supporting Information. Concentrated solutions are nonideal, which results in boundary sharpening,25 so that the large value of f/f° obtained from fit for the concentrated samples cannot be analyzed in terms of shape factor. For H12-Lac solutions, the noninteracting species analysis of Sedfit was also applied. This analysis evaluates, for each species, independent values of s and D. The analysis in terms of hybrid discrete/continuous model of the software sedphat (freely available at www.analyticalultracentrifugation.com) can in principle provide independent values of s and D, and thus Mb, for a main species in the presence of contaminants.26 In view of the heterogeneity of the samples, this approach however did not provide reliable Mb values for solubilized b6 f. Estimates of Bound Surfactant. Svedberg’s equation relates the sedimentation coefficient to the buoyant molecular mass of the particle (Mb), its Stokes radius (RS), the viscosity of the solvent (η), and Avogadro’s number (NA) (22) Gohon, Y.; Pavlov, G.; Timmins, P.; Tribet, C.; Popot J.-L.; Ebel, C. Anal. Biochem. 2004, 334, 318-34. (23) Salvay, A.; Ebel, C. Prog. Colloid Polym. Sci. 2006, 131, 74-82. (24) Dam, J.; Schuck, P. Methods Enzymol. 2004, 384, 185-212. (25) Solovyova, A.; Schuck, P.; Costenaro L.; Ebel. C. Biophys. J. 2001 81, 1868-80. (26) Schuck, P. In Modern Analytical Ultracentrifugation: Techniques and Methods; Scott, D. J., Harding, S. E., Rowe, A. J., Eds.; The Royal Society of Chemistry Publishing: Cambridge, U.K., 2005; pp 26-50.

8884 Langmuir, Vol. 22, No. 21, 2006 s ) Mb/NA6πηRS

Lebaupain et al. (1)

Stokes radii of 49 and 66 Å for the b6 f monomer and dimer in DDM, respectively, were determined by calibrated size exclusion chromatography using a SD200 column (Pharmacia) equilibrated in 20 mM Tris, pH 8.0, 0.3 mM DDM, 250 mM NaCl, as described in ref 27. The number of surfactant molecules (Nsurf) associated to the b6 f was determined from Mb ) Mb,b6f + Mb,surf ) Mb6f (1 - F νjb6f) + Msurf Nsurf (1 - F°νjsurf) (2) This equation was first used to determine the contribution of the b6 f complex Mb,b6f, including lipids and cofactors but without surfactant, in the case of the dimeric b6 f/DDM complex, for which the amount of bound surfactant NDDM has been determined at low [14C] DDM concentration.5 This value, or a value proportional to the polypeptide content in the case of monomeric b6 f, was re-injected in the same equation to determine Nsurf in other conditions. Msurf and νjsurf are the molar mass and partial specific volume of the surfactant. Circular Dichroism (CD). CD measurements were carried out on a Jobin Yvon CD6 spectropolarimeter, at room temperature, using quartz cells of 0.1 cm optical path. The spectra were recorded between 195 and 260 nm with intervals of 1 nm, integration times of 2 s and a constant band-pass of 2 nm. Molecular Dynamics. Model. All atoms of the F-Lac molecules were explicitly included, except for hydrogen atoms bound to carbons, which were treated as united atoms (CH and CH2). The force-field used was derived from the Gromos96 one with variations in the partial charges according to semiempirical (MOPAC software using the MNDO Hamiltonian29) and ab initio calculations (PC-Gamess software,30 using first a geometry optimization at the SCF level, 6-31G basis set, followed by a single-point charge density calculation including electronic correlations at the MP2 level with the same basis set). In the same way, geometry optimization of the starting structures was performed using semiempirical calculations (MOPAC-MNDO Hamiltonian). Water was modeled according to the single point charge (SPC) model. System. The system consisted of 64 F-Lac molecules randomly oriented and positioned in a cubic simulation box and hydrated by 9237 SPC water molecules. Simulation. The 3.2.1 version of the Gromacs package31,32 was used on a single PC workstation (iP4 2,26 GHz, 512 Mo) running Linux RedHat 9.0 at a rate of approximately 49 ps/CPU hour. The system was first energy-minimized using a steepest-descent method. The simulation box was coupled to an external heat bath at 300 K and to an isotropic pressure of 1 atm using Berendsen’s weakcoupling schemes. Periodic boundary conditions were applied in all three dimensions. A first simulation without any restraints was run for 50 ps in order to adjust automatically the size of the simulation box. After equilibration, the volume was 339.193 nm3, corresponding to an average density of 1034 g‚L-1. The production run was then performed for a total of 15 ns.

Results Synthesis. The synthesis of alkylglucosides of defined structure is generally complex.33,34 Difficulties arise in particular in the control of the anomeric configurations of the saccharidic units, which is greatly responsible for the aqueous behavior of the (27) Harlan, J. E.; Picot, D.; Loll, P. J.; Garavito, R. M. Anal. Biochem. 1995, 224, 557-563. (28) Salvay, A.; Ebel, C. Prog. Colloid Polym. Sci. 2006, 131, 74-82. (29) Dewar, M. J. S.; Thiel, W. J. Am. Chem. Soc. 1977, 99, 4899-4907. (30) Schmidt, M. W.; Baldridge, K. K.; Boatz, J. A.; Elbert, S. T.; Gordon, M. S.; Jensen, J. H.; Koseki, S.; Matsunaga, N.; Nguyen, K. A.; Su, S.; Windus, T. L.; Dupuis, M.; Montgomery, J. A., Jr. J. Comput. Chem. 1993, 14, 13471363. (31) Lindahl, E.; Hess, B.; van der Spoel, D. J. Mol. Mod. 2001, 7, 306-317. (32) http://www.gromacs.org. (33) Paulsen, H. Angew. Chem., Int. Ed. Engl. 1982, 21, 155. (34) Schmidt, R. R. Angew. Chem., Int. Ed. Engl. 1986, 25, 212-235.

Table 1. Physical-Chemical Properties of Lactobionamide-Derived Surfactants at 25 °C short name

H10-Lac

C10H21497.6 1.32a 1.6 ( 0.2 γcmc (mN‚m-1) 33a, 28 ( 1 A (nm2) 0.40a 0.38 ( 0.04 log k′W 3.4 νj (mL‚g-1) n.d. ∆210 (M-1‚cm-1) n.d. hydrophobic tail M (Da) CMC (mM)

H12-Lac

F-Lac

HF-Lac

C12H25525.6 0.25a 0.25 (0.03b 35a 0.39a

C6F13C2H4- C2H5C6F12C2H4703.4 713.5 1.5 ( 0.2 1.0 ( 0.2

4.5 0.82 1.46

3.5 0.59 1.59

21 ( 1 25 ( 1 0.40 ( 0.04 0.54 ( 0.04 3.4 0.62 1.56

a Data from ref 42. b Data from fluorimetry, this work. n.d., not determined.

surfactants. To overcome this problem, multistep syntheses are often required. These include the use of adequate reagents to activate the anomeric center and suitable blocking groups to protect hydroxyls. The O-fluoroalkyl glycosides, having one to three methylene groups between the oxygen and the perfluoroalkyl chain, cannot be obtained following these methods. The most common compounds, with three methylenes, are usually prepared by radical condensation of perfluoroalkyl iodide on an alkyl glycoside in the presence of sodium dithionite,35 while perfluoroalkyl glycosides can be prepared following a Mitsunobu reaction.36 To circumvent the difficulties of multistep syntheses, we adapted the procedure used by Williams and colleagues to obtain glycosylamide-like surfactants.14 This procedure does not require any protection of hydroxyls, since it involves the condensation of an aliphatic amine onto a lactone obtained by dehydration of an appropriate glycosidic acid. The first requirement for a surfactant to being useful in biochemistry is that it should be water soluble. Hydrocarbon surfactants derived from monosaccharide gluconolactone exhibit a poor solubility in water37-39 and their fluorocarbon analogues are insoluble.40 Thus, we chose to introduce a more hydrophilic polar head such as a disaccharide to increase water solubility and selected lactobionolactone derivatives as reasonable targets. The general synthesis pathways are described in Scheme 1. Scaling up was straightforward for hydrocarbon (H10- and H12Lac) and fluorocarbon (F-Lac) molecules. The hemifluorinated surfactant, HF-Lac, was isolated after HPLC purification with 20% overall yield. Physical-Chemical Characterization of the Surfactants. H10Lac, F-Lac, and HF-Lac are very soluble in water (up to at least 50 mM F-Lac and 100 mM HF-Lac). Their CMCs, measured by tensiometry at 25 °C, are given in Table 1. H12-Lac is poorly soluble (5× (H10-Lac and H12-Lac have CMCs of 1.6 mM and 0.25 mM, respectively). Fluorocarbon surfactants are known to exhibit CMCs lower than their hydrocarbon analogues.46 Indeed, F-Lac, with an 8-carbon C6F13C2H4- hydrophobic chain, has a CMC of 1.5 mM, similar to that of the 10-carbon H10-Lac. HF-Lac, however, does not follow this rule: despite its 10-carbon chain (C2H5C6F12C2H4-), its CMC (1.0 mM) is close to that of the 8-carbon F-Lac (Table 1). A similar observation has been made previously regarding the F-TAC and HF-TAC telomers.9 Tensiometric measurements also yield estimates of the limit surface tension (γcmc) and area per surfactant molecule (A; Table 1). For a given chain length, the value of γcmc decreases with increasing fluorine content, as expected.46,47 On the other hand, whereas A is similar for H10-Lac, H12-Lac, and F-Lac (around 0.40 nm2), it is significantly higher in the case of HF-Lac (0.54 nm2). Table 1 also reports values of log k′w, a parameter closely related to the molecule’s water/octanol partition coefficient, which can be obtained from reverse phase HPLC.20,21 Dynamic Light Scattering. Whereas the H-Lac compounds, at concentrations higher than their CMC, assemble into particles with apparent hydrodynamic diameters 8 nm at 2 mM (Table 2). Considering the size of the hydrophobic chain, this value largely exceeds that expected for spherical micelles. In addition, the apparent size of the aggregates increases from 8 to 14 nm when HF-Lac concentration is raised from 2 to 20 mM. At any given concentration, the apparent diameter of the aggregates remained stable over at least a week after the solutions were prepared (Table 2). Apparent Partial Specific Volumes from Density Measurements. As expected, when the fluor content of the hydrophobic chain is increased, νj decreases significantly (Table 1), i.e., the density Fsurf of the surfactant increases. Assuming Fsurf ) 1/νj, surfactant densities of 1.22, 1.62, and 1.69 g‚mL-1 are derived for H12-Lac, HF-Lac, and F-Lac, respectively. These values apply for the micellar surfactant, for measurements were made above their CMC. There was no indication of νj dependency with (42) Syper, L.; Wilk, K. A.; Sokolowski, A.; Burczyk, B. Prog. Colloid Polym. Sci. 1832, 110, 199-203. (43) Dupuy, C.; Auvray, X.; Petipas, C.; Anthore, R.; Rico-Lattes, I.; Lattes A. Langmuir 1998, 14, 91-97. (44) Kalyanasundaram, K.; Thomas, J. K. J. Am. Chem. Soc. 1977, 99, 20392044. (45) Winnik, F. M.; Regismond, S. T. A. Colloids Surf., A 1996, 118, 1-39. (46) Kissa, E. Fluorinated Surfactants: Synthesis, Properties, Applications; Surfactants Science Series 50; Dekker: New York, 1994; Chapter 7, pp 264282. (47) Kissa, E. in Fluorinated Surfactants: Synthesis, Properties, Applications; Surfactants Science Series 50; Dekker: New York, 1994; Chapter 4, pp 92-161. (48) Zana, R. E. Colloids Surf. A 1997, 123-124, 27-35.

surfactant concentrations, up to 10 mg‚mL-1, despite the changes in micelle size observed for HF-Lac and F-Lac (see below). Sedimentation Velocity of H12-Lac. The behavior of H12-Lac at different concentrations was investigated by analytical ultracentrifugation (AUC). A c(s) analysis provides a description of the solution in terms of a particle distribution. For H12-Lac at 2.5 g‚L-1 (4.7 mM), a major peak is observed, which we attribute to the detergent micelle, the signal below 0.04 S, hardly distinguishable, being likely due to the monomer (Figure 1, panels 1A-1C). This behavior does not significantly change from 1.4 to 19 mM, (Figure 1, panel 1D). The value of the sedimentation coefficient of the micelle decreases from 2.8 to 1.8 S when increasing the detergent concentration, which can be related to excluded volume effects, with a limiting value at infinite dilution of 2.8 ( 0.1 S. Because the sample is homogeneous, the sedimentation velocity traces can be analyzed using an algorithm developed for noninteracting particles (see Materials and Methods). Considering the solution composed of two types of particles, monomers and micelles, this allows independent calculations of the sedimentation coefficient s and the apparent diffusion coefficient Dapp for the micelle. From the set of five concentrations in the range 1-10 mg‚mL-1, extrapolation of s and Dapp at infinite dilution provides a hydrodynamic radius of 3.3 ( 0.2 nm (Stokes-Einstein’s equation) and a molecular weight of 57 ( 4 kDa (eq 1 and 2), from which are derived a frictional ratio of 1.25. Given the molecular mass of the H-Lac monomer, a micelle of H12-Lac therefore is a globular assembly of 109 ( 9 molecules, a value that compares well with the aggregation numbers of most detergents.1,49 Sedimentation Velocity of HF-Lac and F-Lac at 2.5 g‚L-1. The hydrodynamic properties of HF-Lac and F-Lac are markedly different from those of H12-Lac. In the top panels (A) of Figure 1 are compared the sedimentation profiles of the three surfactants, at 2.5 g‚L-1, for centrifugation times up to 4 h. The complex sedimentation profiles of HF-Lac and F-Lac indicate that they assemble into heterogeneous macromolecular assemblies, some of which sediment much more rapidly than H12-Lac micelles do. Analyses of the sedimentation profiles in terms of c(s) distribution are shown in Figure 1, panels C1-3. Both fluorinated surfactants exhibit a component with s < 2.5 S, i.e. roughly similar to H12Lac micelles, along with larger assemblies with heterogeneous s values in the ranges 5-17 S for HF-Lac and 5-10 S for F-Lac (note that only the general envelope of the distributions, and not the details, is relevant here, since the c(s) analysis assumes the solution to be comprised of noninteracting particles of similar shape, which is probably not the case here -see below). The component with a small s can reflect either the presence of small micelles at equilibrium with larger assemblies or the dissociation of the latter in the ultracentrifuge due to the formation of a gradient of concentration. Sedimentation Velocity of HF-Lac and F-Lac at Various Surfactant Concentrations. Figure 1 (panels D1-3) shows the superimposition of c(s) distributions, for each of the three surfactants, at concentrations ranging from typically 1 to 10 g‚L-1 (1.4-19 mM). Qualitatively, as at 2.5 g‚L-1, two types of macromolecular assemblies are evidenced, with s values either smaller or larger than 5 S. The mean s-values for HF-Lac and F-Lac assemblies increase with the concentration. At the highest concentrations of HF-Lac (26 and 38 mM), phase separation at the bottom of the cell is suggested by the loss of a clear interference pattern, but the species in solution appear rather homogeneous, with a main component in the c(s) analysis at 17-19 S. Thus, (49) Neugebauer, J. A guide to the properties and uses of detergents in biology and biochemistry; Calbiochem Co.: La Jolla, CA, 1988.

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Figure 1. Sedimentation velocity at 20 °C of (1) H12-Lac, (2) HF-Lac and (3) F-Lac. Panels A. Selection of experimental (dots) and fitted (lines) profiles, corrected for systematic errors, at 2.5 g‚L-1 surfactant (i.e. 4.7, 3.5, and 3.6 mM, respectively). In each case, the last profile corresponds to 4 h of sedimentation at 42 000 rpm. B. Differences between experimental data and fitted curves. C. Corresponding c(s) distribution. D. Superimposition of c(s) distributions for concentrations in the range 1-10 g‚L-1 (1.4-19 mM), with additional concentrations of 25.6 (*) and 38.4 mM (**) for HF-Lac; for clarity, the latter two curves have been rescaled to an equivalent path length and divided by a factor of 16.

the size of the assemblies stops increasing significantly only at [HF-Lac] > 20 mM. Circular Dichroism. The chirality of supramolecular assemblies was investigated by CD spectroscopy in the 0.16-9 g‚L-1 range (0.2-12.6 mM). The general aspect of the spectra of H12-Lac, HF-Lac, and F-Lac solutions was similar, and it did not change significantly with concentration. The maximum intensity of the CD signal was proportional to concentration, without any indication of a break at the CMC (see the Supporting Information). ∆210 differed by 15 h), protein/surfactant complexes have sedimented at the bottom of the cell. When the pellet of b6 f in either 2 or 5.5 mM HF-Lac is resuspended and re-subjected to (50) Pierre, Y.; Breyton, C.; Robert, B.; Vernotte, C.; Popot, J.-L. J. Biol. Chem. 1997, 272, 21901-21908.

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Figure 4. Sedimentation velocity analysis of the b6 f complex in 0.2 (A) or 5 mM (B) DDM and 2 (C) or 5.5 mM (D) HF-Lac at 4 °C. The distribution of the b6 f complex is monitored at 280 (solid line) and 420 nm (dotted line), that of the total material (including the surfactant) by the interference signal (dashed line). Curves have been normalized to a value of 1 for the peak of dimer (A, C, and D), except in B where the 280 and 420-nm curves were normalized to a value of 1 for the peak of monomer and interference data are in arbitrary units. m, free surfactant micelles; M, b6 f monomer; D, b6 f dimer.

Figure 5. Sedimentation analysis of the b6 f complex on 10-30% sucrose gradients in the presence of 0.2 and 5 mM DDM, 0.3 and 5 mM H12-Lac, 2 and 5.5 mM HF-Lac, 2 and 5 mM F-Lac. Gradients were centrifuged 4 h at 200 000 × g. D, dimer; M, monomer.

AUC, essentially the same profiles are obtained as in the first run (not shown). This suggests that the protein does not aggregate nor dissociate, even when highly concentrated and in the presence of high HF-Lac concentrations. On the contrary, resuspension of the b6 f centrifuged in the presence of 0.2 mM DDM resulted in an increase of the monomer/dimer ratio as compared to that in the first run (not shown). The sedimentation behavior of b6 f/HF-Lac complexes was also examined by centrifugation in sucrose gradients, a standard approach to fractionating protein mixtures (see, e.g., ref 5). In keeping with AUC observations, the b6 f migrated faster in gradients containing either HF- or F-Lac than it did in the presence of either DDM or H12-Lac (Figure 5). Moreover, the protective effect of the fluorinated tails was again observed: in the presence of low concentrations of DDM or H12-Lac (0.2 and 0.3 mM, respectively), of HF-Lac (either 2 or 5.5 mM), or of F-Lac (2 or 5 mM), the b6 f migrated as a dimer (Figure 5, band D). High detergent concentrations (5 mM DDM or 5 mM H12-Lac), on the other hand, initiated the dissociation of the complex, as evidenced by the appearance of an upper band of monomer (Figure 5, band M). Estimating the Mass of Bound Surfactant. The amount of detergent bound to the b6 f dimer in the presence of low concentrations of [14C] DDM has been determined previously to be ∼260 molecules per dimer.5 By combining this information with the sedimentation coefficient (from AUC) and the Stokes

Figure 6. Evolution over time of the enzymatic activity of cytochrome b6 f upon incubation in four different surfactants either (A) at a concentration close to the CMC or (B) at a higher concentration. Samples were kept on ice in the dark. Activity is monitored immediately after dilution of catalytic amounts of b6 f in 0.3 mM DDM, by following spectroscopically the reduction of plastocyanin, the electron acceptor of the complex, in the presence of plastoquinol, its electron donor.18

radius (from SEC) determined in the presence of DDM, it is possible (from eqs 1 and 2; see Materials & Methods) to calculate the contribution to the buoyant molar mass of the b6 f dimer, including cofactors and lipids but without the detergent, to be

Characterization of Lactobionamide Surfactants

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Table 3. Determination of the Amount of b6 f-Bound Surfactant, Nsurf 0.2 and 5 mM DDM s (S) 4 °C Mb (kDa)a Nsurfb (molecules per complex)

dimer

monomer

7.2 87 260*

4.8 43 200

2 mM HF-Lac

5.5 mM HF-Lac

dimer

dimer

monomer

9.7 120 200

11.0 130 260

8.0 70 170

a The buoyant molar mass of the b6 f/surfactant complex, Mb, was determined from the sedimentation coefficient s and the Stokes radius of the protein obtained in DDM, using Svedberg’s equation (eq 1).b Nsurf was determined using eq 2, taking as a reference Nsurf for the b6 f dimer at low DDM concentration (*), determined using [14C]DDM (from ref 5), and considering that b6 f binds only HF-Lac and no DDM molecules (see Materials & Methods and Results).

Mb,b6f,D ) 62.7 kDa. For the monomer, which loses two proteic subunits and most lipids,5 we can assume a value proportional to the polypeptide content (Mb,b6f,M ) 24.4 kDa), the loss of lipids being essentially without effect on the buoyant mass since they are nearly density-matched in water. Knowing the νj of each surfactant and assuming the Stokes radius (RS) of the complexes to be similar in DDM and HF-Lac, eq 2 yields, for each b6 f/ surfactant species, an estimate of the mass of bound surfactant (Table 3). Not surprisingly, the monomeric b6 f is calculated to bind less DDM than the dimer, in keeping with its smaller hydrophobic cross-section (10 transmembrane R-helices vs 24 for the dimer). The calculated amount of bound surfactant is similar for the b6 f dimer in 5.5 mM HF-Lac and in DDM (a confirmation would require a precise measurement of the RS of the complex in HF-Lac, which is practically difficult because of the large volumes of solution it would require). When increasing the DDM concentration from 0.2 to 5 mM, the values of the sedimentation coefficients of the monomer or dimer (and derived estimations of bound detergent) do not change. Such is not the case for the b6 f in HF-Lac: the sedimentation coefficient for the dimer rises from 9.7 to 11 S when HF-Lac concentration increases from 2 to 5.5 mM. This may reflect either an increase of surfactant binding, due to the tendency of the surfactant itself to form larger aggregates as the concentration rises, or perhaps simply the fact that DDM was not totally removed (see Materials & Methods), a small fraction of it remaining associated to the protein at low HF-Lac concentrations, whereas it would be diluted into the micellar phase at the higher HF-Lac concentration. Protein Stability in DDM, H12-Lac, HF-Lac, and F-Lac Solutions. To assess the stability of the complex, b6 f purified in 0.2 mM DDM was transferred to solutions of the different surfactants, at two different concentrations, by centrifugation in sucrose gradients. The enzymatic activity of the band corresponding to the dimer was then measured as a function of storage time (Figure 6). At surfactant concentrations close to the CMC, day 0 activities are relatively similar. Upon extended incubation at 4 °C, HF- and F-Lac preserved the activity quite efficiently, as well as 0.2 mM DDM. H12-Lac at 0.3 mM, on the other hand, appeared more inactivating (Figure 6A), with, however, quite some variability from one experiment to the next (not shown). Increasing the concentration of either H12-Lac or DDM well above their CMC dramatically destabilized the b6 f, which was totally inactivated after only 7 days of incubation. This is consistent with the high proportion of monomer observed in the sucrose gradients under these conditions (Figure 5). Inactivation was significantly slower at high HF-Lac or F-Lac concentrations (Figure 6, panels A and B), in keeping with the protective effect

of substituting a hydrogenated with a fluorinated or a hemifluorinated tail previously observed with F- and HF-TAC.10

Discussion Physical-Chemistry of Lactobionamide Surfactants. Differences between F-Lac and the two H-Lac compounds conform to previous observations on other series of surfactants with either perfluorinated or hydrocarbon hydrophobic moieties (see, e.g., refs 47 and 51): namely, the substitution of fluorine to hydrogen results, for a given chain length, in lowering both the CMC and the limit surface tension, γcmc. The first phenomenon is due to the absolute value of the free energy of micellization being larger, at constant chain length, for fluorinated compounds, which has been correlated with the larger volume and surface of the cavities created in water by fluorinated chains (see, e.g., ref 52). The second indicates that the energy of transfer to the air/water interface becomes even more favorable, with the result that, at the CMC, the pressure in the film is higher. The parameter log k′w is usually taken to reflect the “hydrophobic character” of hydrocarbon surfactants, and, indeed, H10-Lac exhibits a lower log k′w than H12-Lac, which is more hydrophobic. However, the interpretation of log k′w measurements is more complex for fluorinated surfactants, for they reflect the partition of the molecule between a hydrocarbon C18 stationary phase and the polar mobile phase. The apparently paradoxical behavior of F-Lac, which, despite its lyophobicity, appears to have as high an affinity for the C18 medium as its longer-chain hydrogenated counterpart H10-Lac, will be discussed elsewhere. The behavior of HF-Lac in solution is atypical, and departs from that of H- or F-Lac in several important respects: (i) The CMC of HF-Lac is similar to that of F-Lac despite the presence of an additional ethyl group. A similar observation has been made previously for HF- and F-TAC.9 It differs from the usual behavior of hydrocarbon detergents, whose CMC, as a rule, diminishes by a factor of 5-10 upon addition of an ethyl group (as indeed observed for H12-Lac and H10-Lac). (ii) Whereas the same area per surfactant molecule (A) would be expected for the same hydrophilic head (as is observed for H10-, H12-, and F-Lac, Table 1), the value obtained for HF-Lac is markedly higher (0.54 nm2 instead of ∼ 0.4 nm2). (iii) Whereas the limit surface tension measured for F-Lac is lower than that for H10- and H12-Lac, as expected for a fluorinated surfactant,47 an intermediate value is obtained for HF-Lac (Table 1). (iv) Finally, F-Lac and HF-Lac have similar values of log k′w, despite the presence of an additional ethyl group on HF-Lac. The effect of adding an ethyl tip at the end of a fluorinated chain on the energetics of micellization strongly differs from that of elongating an alkyl chain by two carbons. This is best illustrated by the fact that the CMC of HF-Lac, rather than dropping by about a decade, remains more or less the same as that of F-Lac (Table 1). Among the contributions that more or less cancel the increase of hydrophobicity expected from adding the ethyl tip, two are likely to be i) poorer packing, related either to unfavorable van der Waals interactions between fluorinated and hydrogenated groups and/or to steric hindrance in the core of HF-Lac micelles, and ii) the acidic character of the methylene group vicinal to the fluorocarbon core -a consequence of the electron withdrawing effect of fluorine-, which favors hydrogen bonding with the aqueous phase. Such an effect would increase the energy of transfer of the hemifluorinated chain of HF-Lac (51) Barthe´le´my, P.; Cuvillier, N.; Chaudier, Y.; Benattar, J.-J.; Pucci, B. J. Fluorine Chem. 2000, 105, 95-102. (52) Ravey, J. C.; Ste´be´, M. J. Colloids Surf. A 1994, 84, 11-31.

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to a hydrophobic phase and shift its partition coefficient toward water. The same two features are likely to contribute to the higher values of the limit surface tension, γcmc, and the area per surfactant molecule, A, for HF-Lac as compared to F-Lac. This behavior is reminiscent of that observed when replacing a single fluorine atom with a hydrogen one at the tip of the perfluorinated chain of nonionic surfactants, which also results in raising their CMC, γcmc, and A.52,53 Probably because of the same underlying mechanisms, stable Newton black films can be formed from either hydrogenated or fluorinated analogues of H- and F-TAC, but not from hemifluorinated ones.51 The aggregation behavior of the surfactants is also markedly different. H12-Lac forms homogeneous micelles of ∼100 molecules, as is frequently observed with hydrogenated detergents.1,49 On the contrary, HF- and F-Lac self-associate into large assemblies, whose size increases with the concentration of surfactant. This was observed both by analytical ultracentrifugation (sedimentation coefficients between 10 and 20 S, vs ∼2 S for H12-Lac micelles; Figure 1) and, for HF-Lac, by dynamic light scattering (apparent hydrodynamic diameter 8-14 nm). The latter figure is much larger than that expected for a spherical micelle, which is usually comprised between 3 and 6 nm. The molecular packing parameter concept proposed by Israelachvili et al.,54 V0/al0 (where V0 and l0 are the volume and length of the surfactant tail and a the surface area per molecule, defined at the hydrocarbon-water interface and assimilated, in a first approximation, to the surface area of the hydrophilic head), emphasizes the importance of a in predicting the shape and size of equilibrium aggregates. However, as shown by Nagarajan,55 the properties of the tail also have a controlling role, and exert a strong influence on the tendency of the surfactant to form spherical micelles, rodlike micelles, or lamellar phases. Within the concept of molecular packing parameter, “the area per molecule is not a variable connected to the geometrical shape and size of the surfactant headgroup, but rather a thermodynamic quantity obtained from equilibrium considerations of minimum free energy”.55 The interactions between the hydrocarbon tip and the fluorocarbon core of HF-Lac surfactants therefore may influence the packing of the hydrophobic tail, the type of aggregates formed, and the energy of micellization. The large aggregates observed for F- and HF-Lac could be rodlike or disk assemblies. This is confirmed by molecular dynamics simulations, which suggest that F-Lac has a propensity to form cylindrical micelles. Such a behavior has already been reported for ammonium perfluorooctanoate and cationic or polyoxyethylenederived fluorosurfactants.46,52,56,57 In general, for a given type of polar head, it seems that fluorocarbon surfactants exhibit a (53) Eastoe, J.; Paul, A.; Rankin, A.; Wat, R. Langmuir 2001, 17, 7873-7878. (54) Israelachvili, J.; Mitchell, D.; Ninham, B. J. Biochim. Biophys. Acta 1977, 470, 185-201. (55) Nagarajan, R. Langmuir 2002, 18, 31-38. (56) Burkitt, S. J.; Ottewill, R. H.; Hayter, J. B.; Ingram, B. T. Colloid Polym. Sci. 1987, 265, 619-627.

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propensity to form structures with less interfacial curvature than hydrocarbon surfactants. Protective Effect of the Fluorinated Tail in Biochemistry. From the point of view of the biochemist, a key point in the evaluation of novel surfactants is an examination of their capacity to keep membrane proteins under a water-soluble, homogeneous, active and stable form. This study was carried out by comparing the behavior of a particularly fragile membrane protein complex, cytochrome b6 f, in DDM, the most stabilizing detergent for this protein, and in the three types of lactobionamide surfactants. At a surfactant concentration close to the CMC, the b6 f complex is homogeneous and dimeric in HF-Lac, whereas it shows some dissociation into inactive monomers in DDM. However, the rate at which the enzymatic activity decays over time is similar in both surfactants. Upon increasing the concentration of surfactant, i.e. in the presence of a noticeable concentration of free micelles, the stabilization of the dimer by HF-Lac becomes obvious, as evidenced by AUC, sucrose gradients and activity measurements. F-Lac shows the same capacity to stabilize the dimer and preserve the enzymatic activity. The fact that the b6 f complex in HF-Lac neither monomerizes nor aggregates when centrifuged at the bottom of the AUC cell (corresponding to extreme concentrations) is an interesting observation, which suggests that hemifluorinated surfactants may be considered for structural biology applications (such as NMR or X-ray crystallography) that necessitate highly concentrated protein. Another interesting result is, paradoxically, the poor capacity of H12-Lac to preserve b6 f activity over time. This shows that the lactobionamide head is not particularly favorable to protein stability, which leaves open the perspective of optimizing tail/head combinations so as to obtain yet milder fluorinated or hemifluorinated surfactants. Acknowledgment. We are grateful to D. Picot for the determination of the Stokes radius of b6 f/DDM complexes and for very useful discussions and to E. Rousselet for preliminary work on HF-Lac in biochemistry. This work was supported by the C.N.R.S., the C.E.A., the Universities of Paris-7, Avignon et Pays du Vaucluse, and Joseph Fourier (Grenoble, France), and by E.C. Specific Targeted Research Project “Innovative tools for membrane structural proteomics”. F.L. , B.O. and N.M. were recipients of fellowships from the “Ministe`re de l’Enseignement Supe´rieur et de la Recherche”. A.S. was supported by a CNRS fellowship (“poste rouge”). Supporting Information Available: Parameters used in the c(s) analysis of sedimentation velocity. Tensionetric curves of F-Lac and HF-Lac (Figure S1). Spectrofluorimetric measurements of H12-Lac CMC (Figure S2). Circular dichroism of HF-Lac, H12-Lac, and F-Lac (Figure S3). This material is available free of charge via the Internet at http://pubs.acs.org. LA061083L (57) Wang, K.; Karlsson, G.; Almgren, M.; Asakawa, T. J. Phys. Chem. B 1999, 103, 9237-9246 and references cited.