Stabilization of Hydrophobic Colloidal Dispersions in Water with

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Stabilization of Hydrophobic Colloidal Dispersions in Water with Amphiphilic Polymers: Application to Integral Membrane Proteins C. Tribet,*,† R. Audebert,†,‡ and J.-L. Popot§ Laboratoire de Physico-Chimie Macromole´ culaire, Universite´ Paris 6, CNRS URA 278, ESPCI, 10 rue Vauquelin, 75231 Paris Cedex 05, France, and Institut de Biologie Physico-Chimique and Universite´ Paris 7, CNRS UPR 9052, 13 rue P. et M. Curie, 75005 Paris, France Received February 10, 1997. In Final Form: August 4, 1997X

Stabilization of aqueous dispersions of highly hydrophobic colloidal particlesshere three integral membrane proteins of nanometric dimensionsswere obtained by substituting the classical protective layer of surfactant with low molecular weight amphiphilic polymers (“amphipols”). These polymers, derived from poly(acrylic acid), adsorb onto the particle despite the presence of residual surfactant from the initial stock solutions of proteins. Unbound amphipols and surfactants can be subsequently removed without dissociation of the complex or precipitation. As a consequence of multipoint attachment between the partners, complexation appears irreversible on the time scale of a few hours to a few days. Protein/ amphipol complexes contain a much lower number of alkyl groups per particle as compared with classical protein/surfactant complexes. The density of coverage (≈100-200 alkyl groups per particle) increases with the salt concentration of the medium but is not sensitive to pH.

Introduction The stabilization of a dispersion of colloidal particles in a fluid involves the control of numerous antagonistic forces in order to prevent coagulation. In these systems, aggregation processes can be controlled by modifying interfaces with adsorbed agents like surfactants or polymers.1 The resulting introduction of steric barriers or modification of the Coulombic effect, thanks to charged additives, can deeply modify the interactions between particles. In water, the hydrophobic effect also has to be taken into account, especially between organic particles (latex, globular proteins). In general, masking hydrophobic surfaces with surfactants can stabilize the suspensions for days.2 The composition of such complex mixturessfree monomeric surfactant, micelles, and “decorated” colloidal particlessand the possible presence of other organized structures strongly depend on such parameters as the concentrations, the temperature, etc. We have endeavored to develop hydrosoluble polymeric agents that can stick to hydrophobic surfaces. From multisite physisorption of the macromolecules onto hydrophobic colloids, we expect a dramatic decrease of the kinetics of desorption and an increase in the energy of association as compared with monomeric surfactants. Such compounds ought to combine the versatility of surfactant layers, which can adapt to surfaces of various roughness and shapes, with the advantages of quasi-irreversible association. In particular, the stability of the dispersions should not depend on the presence of free polymer. We have synthesized amphiphilic polymers of low molecular weight (Mw < 34 000; “amphipols” in the following). They †

CNRS URA 278. This paper is dedicated to the memory of Roland Audebert who died while the proofs were being corrected. He initiated our research on protein/polymer interactions. Through his faith and determination, he greatly contributed to launching and developing this project. § CNRS UPR 9052. X Abstract published in Advance ACS Abstracts, September 15, 1997. ‡

(1) Ottewill, R. H. In Scientific Methods for the Study of Polymer Colloids and their Applications; Candau, F., Ottewill, R. H., Eds.; Kluwer Academic Publisher: London, 1990; pp 129-157. (2) Tanford, C. The Hydrophobic Effect: Formation of Micelles and Biological Membranes; John Wiley and Son: New York, 1980; p 233.

S0743-7463(97)00136-4 CCC: $14.00

Figure 1. Structure of amphipols used in the present study. A 8-75 corresponds to a random copolymer (e ) 0); A*8-75 is a terpolymer (with e ) 0.0025). The asterisk indicates the 14C atom.

are random copolymers of acrylic acid, N-octylacrylamide, and N-isopropylacrylamide. They were obtained from commercially available poly(acrylic acid) by forming amide bonds with octylamine or isopropylamine.3 The hydrophobicity of the chains is mainly adjusted by the amount of octyl groups. The charge density of the macromolecule can be modified, at quasi-constant hydrophobicity, by grafting isopropyl groups. In the present paper, we focus on one polymer structure, denoted A 8-75 (A for anionic, 8 referring to the average molecular weight (8000) and 75 to the molar fraction of free carboxylic groups (75%)) (Figure 1). We have examined the ability of amphipols to maintain soluble in water highly hydrophobic membrane proteins. Three purified integral membrane proteins (IMPs) were chosen as model globular particles of nanometric dimensions: the photosynthetic reaction center from Rhodobacter sphaeroides4 R-26 (RC, Mw ≈ 100 000), dimeric cytochrome b6 f from Chlamydomonas reinhardtii5 (Mw ≈ 211 000), and the trimeric matrix porin OmpF from Escherichia coli6 (Mw ≈ 111 000). In vivo, these IMPs span lipid bilayers. IMP structure and composition are not markedly different from those of water-soluble proteins except that transmembrane-segments of polypeptide chains are markedly more hydrophobic. Their surface displays a central hydrophobic beltsthe transmembrane region, about 3 nm in height and a few nanometers in (3) Wang, T. K.; Iliopoulos, I.; Audebert, R. Polym. Bull. 1988, 20, 577. (4) Verme´glio, A. Biochim. Biophys. Acta 1977, 459, 516. (5) Pierre, Y.; Breyton, C.; Kramer, D.; Popot, J.-L. J. Biol. Chem. 1995, 270, 29342. (6) Garavito, R. M.; Rosenbusch, J. P. Methods Enzymol. 1986, 125, 309.

© 1997 American Chemical Society

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diameterscapped by two hydrophilic ends. All of the three IMPs are totally insoluble in water, despite the presence of polar groups and ionizable groups on the hydrophilic caps. Usually, purified IMP dispersions are handled in surfactant solutions near the surfactant’s critical micellar concentration (cmc).7,8 The major interests of IMP in the field of colloids is their monodispersity and their sensitivity to change in their surrounding medium (induced denaturation). In favorable cases, the structure of native IMP can be established in atomic detail (by X-ray diffraction of protein crystals), yielding an exact knowledge of such important surface properties as composition, distribution of charges, or hydrophobic patches, etc. The charges of globular proteins, mainly located on their surface, vary with pH. This phenomenon has previously opened the way to extensive investigations of polyanion/ polycation interactions in mixtures of proteins and synthetic polyelectrolytes. Soluble or insoluble complexes of polyanion/polycation, reversible and irreversible associations have been recognized.9-11 Since the pioneering work of Morawetz9 the main application of this research has been the purification of proteins by precipitation with a polymer carrying an opposite charge. Recently, Sunamoto and colleagues reported the formation of supramolecular assemblies between soluble proteins and selfaggregates of pullulan grafted with cholesterol dangling groups;13a,b these hydrogel nanoparticles can accommodate native globular proteins. The authors suggested two possible origins of the association: a direct participation of the hydrophobic cholesterol groups or hydrogen bond formation with the backbone. Apart from this work, little attention has been paid to the importance of the hydrophobicity of the partners, even though it can play a major role.12-14 In the present paper, we aim at a better understanding of the mechanism of hydrophobically driven complexation between IMP and amphipols, focusing on the composition and the conditions of formation of complexes. Materials and Methods Membrane proteins classically are made water soluble by complexation with surfactants (usually derivatives of sugars or of alkylpoly(oxyethylene)). The surfactants used here are Hecameg (6-O-(N-heptylcarbamoyl)methyl-R-D-glucopyranoside, cmc ≈ 19.5 mM) purchased from Vegatec (Villejuif, France), laurylmaltoside (LM) (cmc ≈ 0.17 mM, from Sigma), and octylPOE (surfactant present in the stock solutions of OmpF, cmc ≈ 0.2%). [14C]-labeled LM was a gift from M. Le Maire (C.E.A., Saclay, France). Solutions were buffered at pH 8 either with a Tricine-NaOH mixture or with an ammonium phosphate (AP)-NaOH buffer (Prolabo, France). Synthesis of [14C]-Labeled Amphipol (“A*8-75”). A*8-75 was synthesized from A 8-75 by further grafting 0.25% of acrylic units with [14C]-labeled n-butylamine (Figure 1). A*8-75 was obtained by a similar synthesis as unlabeled amphipols;3,14 octylgrafted poly(acrylic acid)3 in N-methyl-2-pyrrolidone (Prolabo) (7) Helenius, A.; Simons, K. Biochim. Biophys. Acta 1975, 415, 29. (8) Hartmut, Michel Crystallisation of Membrane Proteins; Hartmut, Michel, Ed.; CRC Press: Boca Ratan, FL, 1990; Chapter 3. (9) Morawetz, H.; Hughes, W. L., Jr. J. Phys. Chem. 1951, 56, 64. (10) Margolin, A. L.; Sherstyuk, S. F.; Izumrudov, V. A.; Zezin, A. B.; Kabanov, A. Eur. J. Biochem. 1985, 146, 625. (11) Dubin, P. L.; Gao, J.; Mattison, K. Sep. Purif. Methods 1994, 23 (1), 1. (12) Petit, F.; Audebert, R.; Iliopoulos, I. Colloid Polym. Sci. 1995, 273, 777. (13) (a) Nishikawa, T.; Akiyoshi, K.; Sunamoto, J. Macromolecules 1994, 27, 7654. (b) Akiyoshi, K.; Nishikawa, T.; Mitsui, I.; Miyata, T.; Kodama, M.; Sunamoto, J. Colloids Surf. 1996, 112 (2/3), 91. (14) Tribet, C.; Audebert, R.; Popot, J.-L. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 15047. (15) Breyton, C.; Tribet, C.; Olive, J.; Recouvreur, M.; Popot, J.-L. Proceedings of the Xth International Meeting on Photosynthesis; Mathis, P., Ed.; Kluwer Academic Publisher: Dordrecht 1995; p 591.

Langmuir, Vol. 13, No. 21, 1997 5571 was poured onto [14C]-labeled n-butylamine (Sigma, 0.5% mol/ mol of the acrylic units in the polymer) previously dried under nitrogen. The coupling reagent N,N′-dicyclohexylcarbodiimide (Jansen Chimica) was added (twice the molar amount of butylamine). After 2 days of reaction at room temperature, the polymer was precipitated by neutralization with MeONa in methanol (5.25 M) and addition of ethanol. The product was further purified by two precipitations in ethanol from concentrated aqueous solutions. The actual degree of n-butyl grafts (0.25% mol/mol) was determined from the β-activity of a 10 mg/ mL polymer stock solution in water. The grafting yield of 50% only is likely to be due to both the two-step grafting (yield of initial octylamine grafting is about 100%) and the presence of alcohol, at trace amounts, from the solution of labeled amine. Isolation of Proteins. Cytochrome b6 f, from C. reinhardtii, was purified from one to a few days before the experiments by a previously reported procedure.5 Composition of the stock solutions was as follows: ≈2.5 µM b6 f native protein particles (non covalent dimers, Mw ≈ 300 000 g/mol including bound surfactant16 ) in Hecameg (20 mM), AP-NaOH buffer (400 mM), egg yolk L-R-phosphatidylcholine (0.1 g/L) (a lipid required to prevent denaturation, from Sigma), protease inhibitors (aminocaproic acid (5 mM), benzamidine (0.2 mM), and phenylmethylsulfonyl fluoride (0.2 mM)). For measurements of bound surfactant, stock solutions of b6 f in LM were prepared from Hecameg solutions by elution on a Sephadex G50 column saturated with LM (0.2 mM) and Tricine-NaOH buffer (20 mM, pH 8). The solution was supplemented with [14C]-LM (final concentration of LM 0.264 mM including 0.064 mM of labeled surfactant) and incubated 30 min before the sedimentation analysis (see below). Purified OmpF porin (a trimer, Mw ≈ 111 000 g/L) was a kind gift from J. P. Rosenbusch (Biozentrum, Basel). Two different preparations were used: OmpF 1.2 or 20 g/L, AP buffer (20 mM pH 7.2), NaCl (0.1 M), octylPOE (1% wt/wt), EDTA (1 mM), sodium azide (3 mM).6 The more concentrated stock solution was diluted 5-fold in AP buffer (100 mM, pH 8) before use. Purified photosynthetic reaction center R-26 monomer (RC) was a kind gift from B. Schoepp (CNRS UPR 9061, Gif sur Yvette, France); initial solution was RC (30 g/L), Hecameg (20 mM), Tricine-NaOH buffer (20 mM, pH 8), potassium chloride (3 mM), and magnesium chloride (3 mM).4 This solution was further purified to remove aggregates by sedimentation (see below) on a 5-20% (wt/wt) sucrose gradient containing Hecameg (20 mM) and Tricine-NaOH buffer (20 mM, pH 8) (final concentrations RC ≈ 7-30 µM and sucrose ≈ 10% wt/wt). All solutions were kept at 4 °C. Measurements of Protein Solubility at Low Concentration of Surfactant. Aliquots from stock solutions of polymer A 8-75 (5-30 g/L) were added to RC solutions (9.6-29 µM) in Hecameg (20 mM, i.e. just above the cmc) and Tricine-NaOH buffer (20 mM, pH 8). The mixtures were then brought to a final 10-fold dilution with amphiphile-free Tricine buffer. After a 10 min incubation at 4 °C, the samples were centrifuged for 30 min at 4 °C in the A-110 rotor of an Airfuge (Beckman) at 20 psi (≈140 kPa, ≈210000g). The protein in the supernatant was titrated by spectrophotometry. A similar procedure was applied with OmpF (at 3.5 µM in the stock solution) and an AP-NaOH buffer (100 mM, pH 8). Sedimentation Analysis. Complexes of IMP and amphipol were obtained at 4 °C by supplementation of 20-100 µL stock solution of IMP with 7-20 µL of polymer in water (5-30 g/L) followed by dilution with buffer (AP or Tricine).14 The surfactant from the protein stock solution reached a final concentration of half the cmc. Portions (100-200 µL) of these mixtures were layered onto 2 mL of 5-20% (wt/wt) linear sucrose (Sigma) density gradients containing the same buffer as the mixtures, -aminocaproic acid (5 mM) and, in some cases, other additives like surfactant, polymer, and, in the case of b6 f, additional protease inhibitors (cf. “isolation of proteins”). The tubes were centrifuged for 5-7 h at 54 000 rpm, 4 °C, in a Beckman TL100 ultracentrifuge (TLS 55 rotor, ≈250000g). Fractions (120 µL) were collected from the top with a Hamilton syringe. The bottom of the tube was washed with 120 µL buffer (“pellet”). The concentration of (16) Breyton, C.; Tribet, C.; Dubacq, J.-P.; Olive, J.; Popot, J.-L. J. Biol. Chem. in press.

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IMP was determined spectrophotometrically from the absorbance at 278 nm (porin), 802 nm (RC), or 554 and 564 nm (redox difference spectrum of b6 f.5,14). In experiments involving A*875, the concentration of the polymer was determined from the β-activity of a 20 µL volume diluted in 5 mL Aqualuma-plus (Lumac LSC, Groningen, The Netherlands) using a LS 1801 liquid scintillation counter (Beckman). Effect of Ionic Strength. Low ionic strength stock solution of IMP contained a 20 mM Tricine-NaOH buffer (obtained, in the case of b6 f, by dialysis overnight of the stock solution against a 20 mM Hecameg, 20 mM Tricine buffer). High ionic strength IMP solutions were either stock solutions of b6 f in 400 mM ammonium phosphate-NaOH buffer or stock solutions of RC or porin supplemented with aliquots of ammonium phosphateNaOH buffer to a final concentration of 100-200 mM. The IMP solutions so obtained were supplemented with a large excess of A*8-75 ((2-10)-fold the final bound quantity, 7-20 µL aliquots) and diluted (to 200 µL) in the appropriate buffer, to a surfactant concentration 2-fold below the cmc. Sedimentation analysis was subsequently carried out as described above.

Results and Discussion Formation of Amphipol/Protein Complexes. We have shown previously, using a dilution procedure, that amphipols and integral membrane proteins form hydrosoluble complexes.14 In the absence of polymer, dilution of the protein stock solutions well below the critical micellar concentration of the surfactant (a tenth to a thirtieth) resulted in the aggregation of proteins, attributed to the removal of most of the surfactant from their hydrophobic transmembrane region. Total precipitation also occurred when IMP solutions were diluted in the presence of poly(acrylic acid), the hydrophilic precursor of amphipols. On the contrary, when supplemented with enough amphipol, IMP stock solutions can be diluted without precipitation of the protein.14 In the present report, we focus on the composition of amphipol-decorated IMP as a model for the study of polymer adsorption onto amphiphilic particles. The amount of amphipol required to keep IMP soluble depends on the protein, but the qualitative behavior is similar for all the IMPs studied to date.14 When increasing concentrations of amphipols were added to IMP stock solutions before dilution with amphiphile-free buffer, two regimes were recognized. Typical results and procedures are shown in Figure 2A for the photosynthetic reaction center. At low amphipol/RC ratios, the soluble fraction of the protein increases sharply with the addition of amphipol; above a threshold ratio, nearly complete solubility of the RC is observed. The critical amphipol/protein ratio required to keep RC soluble seems roughly independent of the initial concentration of the protein as well as of the incubation time before dilution (of the order of a few minutes). However, the boundary between the “high ratio” and the “low ratio” regimes was slightly modified when diluted mixtures were subjected to long incubation times (we observed that the amount of soluble protein, in the low ratio regime, is slightly lower when the precipitation time was increased up to 1 h instead of 10 min, so the boundary between the two regimes is shifted to a higher amphipol concentration). Precipitation is sensitive to the mixing procedure. When supplementation with amphipol and dilution into surfactant-free buffer were carried out simultaneously, by diluting aliquots of IMP stock solution into large volumes of buffered amphipol solutions, the fraction of soluble proteins (after identical incubation and centrifugation times) was much lower than when amphipol addition preceded dilution (Figure 2B). In other words, the aggregation of IMP that follows the loss of their surfactant belt proceeds at a faster rate than their protective

Figure 2. Solubility of membrane protein/amphipol complexes in aqueous solution as a function of the initial polymer/protein ratio. (A) Solubility of RC complexes after amphipol addition in the stock solutions, before final 10× dilution (below cmc of the surfactants). RC initial concentration: b ) 29 µM, 9 ) 19 µM, [ ) 9.6 µM. Dashed lines indicate 100% solubility levels. (B) Fraction of soluble porin (initial concentration 3.5 µM) after either (b) a similar procedure as for A or (9) dilution, before similar incubation/centrifugation procedure, of a small volume of the same stock solution of porin into a large volume of A 8-75, AP-NaOH buffer (100 mM, pH 8).

association with amphipols. This observation strongly suggests that, when amphipols are added to surfactantsolubilized IMP, they associate with it within a few minutes even in the presence of micellized surfactant. Composition of the Complexes. Further experiments on soluble IMP/amphipol complexes relied on the first procedure, namely, supplementation with amphipol prior to dilution. Rate zonal centrifugation is a mild separation technique that can resolve monomers from dimers of IMP as well as separate free amphiphiles (surfactants and amphipols) from IMP complexes. For these experiments, complex formation was initiated by a similar supplementation/dilution procedure as for the solubility tests. However, 2-fold dilution below the cmc of the surfactants was preferred to improve accuracy on the determination of protein final concentration (below 1 µM; experimental uncertainty can reach (10%). In order to determine polymer concentrations, a [14C]-labeled polymer, A*8-75, was used instead of A 8-75 (see Materials and Methods). In sucrose gradients containing no amphiphile, IMP/ amphipol complexes migrate as particles of well-defined size (one sharp zone). We have shown previously that this zone contains native-like IMP, that is, enzymatically active and stable.14 No aggregate (no pellet) is detected provided enough amphipol is present in the mixture layered on the gradient (Figures 3 and 4B). Absolute monodispersity of the complexes cannot be proven by this technique, but the presence of oligomeric aggregates would have been detected. On the other hand, when too little polymer is initially present, part of the protein sediments and forms a large pellet. Aggregation is also clearly

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Figure 3. Sedimentation velocity analysis of a porin (OmpF) /A*8-75 mixture in an amphiphile-free 5-20% sucrose gradient. Details on the procedure are given in the Materials and Methods section. Composition of the initial mixture: OmpF, 0.5 g/L; A*8-75, 2.1 g/L; AP-NaOH buffer, 91.5 mM; NaCl, 50 mM; octylPOE, 0.1%. Centrifugation time 6 h 30 min. Fraction 1 is at the top of the tube, 17 is the resuspended pellet: ([) concentration of A*8-75; (O) concentration of porin; (b) molar ratio of the number of octyl groups belonging to A*8-75 to the number of porin particles in the fractions (assuming the presence of porin trimers only, as shown previously14).

revealed by the presence of protein throughout the lower part of the gradient (Figure 4A). Free amphipol molecules diffuse in the topmost region of the gradient and remain at the top of the tube, while bound amphipols comigrate with the protein and reach the middle of the gradient (Figure 3). On the contrary, the distribution of the surfactant along the gradient does not reveal any comigration (not shown). Indeed, the values of the molar ratio of remaining surfactant to protein in IMP-containing fractions indicate a nearly complete removal of surfactant (for instance, the LM/b6 f dimer mol/mol ratio varies between 12 and 4 in the gradient, while the molar ratio of bound-LM/b6 f in micellar solutions is 260 ( 2015,16). When amphipol/surfactant/IMP mixtures are dialyzed for days against amphiphile-free buffer, most of the surfactant and unbound amphipol cross the membrane without affecting the stability of the dispersed state of the IMP (not shown). As expected for complexes with a defined mean composition, the amphipol/IMP ratio is constant throughout the IMP-containing fractions of the gradients (Figure 3). Even when too little amphipol was present in the mixture at the moment of complex formation, a fraction of the protein remained soluble and sedimented at the same position as complexes prepared with an excess of amphipol (fractions 8-11 on Figure 4). The amphipol/IMP ratio in these fractions was close to that measured when a large excess of amphipol was initially present (Figure 4, top). The average composition of soluble complexes containing IMP therefore appears largely independent of the initial proportions of the partners. It is likely to correspond to amphipol-saturated proteins. Aggregated species are associated with a lower quantity of polymer, as revealed by the decrease of the polymer/IMP ratio from the first IMP containing fraction to the bottom of the gradient

Figure 4. Sedimentation velocity analysis of b6 f /A*8-75 mixtures. (A) Preparation with a subsaturating amount of amphipol. Composition of the initial mixture, layered onto the gradient: b6 f ≈ 2.6 µM; A*8-75, 0.135 g/L; Hecameg, 11 mM; egg phosphatidylcholine, 0.095 g/L; Tricine-NaOH buffer, 20 mM; protease inhibitors (see Materials and Methods). Centrifugation time 5 h 30 min. Fraction 1 is at the top of the tube, fraction 17 is the resuspended pellet. ([) concentration of A*875; (O) concentration of the protein. The concentration in the resuspended pellet (not plotted) was 0.83 µM. Top panel (b): molar ratio of the number of octyl groups belonging to the polymer to the number of protein particles in the fractions (assuming the presence of b6 f dimers only14). (B) Preparation in the presence of a large excess of amphipol. (Same concentrations and centrifugation conditions as above except for [A*875] ) 0.45 g/L in the layered mixture). Top panel (+): molar ratio of octyl groups to b6 f dimers.

(Figure 4A). In conclusion, when a low amount of polymer was added, some IMPs were saturated and remained well dispersed, while others formed aggregates of various sizes containing a smaller proportion of amphipol. Taken together, the results suggest that amphipols and IMP form complexes with a low mass polydispersity. The stability of the complexes despite the removal of free amphipols can be attributed either to a low dissociation rate or to such a high association constant of the partners that trace amounts of free polymer in equilibrium with complexes are sufficient to stabilize them.

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Table 1. Mean Ratio of the Number of Octyl Groups from Bound A*8-75 to the Number of Particlesa gradient content

b6 f

OmpF

RC

no amphiphile A*8-75c A 8-75b Hecameg 20 mM

103 ( 9 n.d.d 98 ( 12 19 ( 6

141 ( 3 157 ( 14 90 ( 6 30 ( 15

112 ( 8 107 ( 6 n.d. n.d.

a Computed by assuming monomers of RC, dimers of b f, and 6 trimers of OmpF in the complexes purified by rate zonal centrifugation at pH 8 (see Materials and Methods and Figure 3 for details on the measurements). Errors correspond to the largest difference between the mean value and the values measured in the 4 major IMP-containing fractions. Buffer composition: Tricine-NaOH 20mM (b6 f ) or ammonium phosphate-NaOH 90mM (OmpF) or 97mM (RC). b [A 8-75] ) 0.05 g/L. c OmpF, [A*8-75] ) 0.045 g/L; RC, [A*8-75] ) 0.023 g/L. d n.d., not determined.

Postconstitutive Modification of the Polymer Layer. The constant value of the amphipol/IMP ratio in amphiphile-free sucrose gradients suggests that the composition of the complexes does not change during their sedimentation in the buffer. This can only be possible if the exchange of bound polymer with the bulk phase is negligible on the time scale of the experiment. In order to examine the reversibility of the complexation, rate zonal sedimentation experiments were carried out in gradients containing amphiphiles. The amount of bound A*8-75 per IMP was measured by centrifugation of the same initial solution (containing IMP, A*8-75, and surfactant below the cmc) in 5-20% gradients containing either (i) buffer only, or (ii) A*8-75, or (iii) A 8-75, or (iv) a low molecular weight surfactant at its cmc. If any exchange with the bulk can occur, different values of the final A*8-75/IMP ratio are expected. In the absence of surfactant in the gradient or in the presence of excess of A*8-75 (conditions i and ii), measured differences remained within experimental uncertainty (Table 1). The presence of free radioactive amphipol in the sedimentation medium therefore did not change the composition of the complexes present in the mixture initially layered on the gradient. Namely, (a) no increase of the amount of adsorbed polymer resulted from the presence of A*8-75 in the gradient, which is an additional proof of the formation of saturated complexes, and (b) no removal of labeled polymer was detected in the absence of free surfactant; therefore, the dissociation of the complex must be either extremely limited or extremely slow. This behavior underlines the high affinity of amphipols for IMP. Indeed it is assumedsand experimentally proven17 sthat the adsorption of a polymer on a colloid can be considered as practically irreversible. However, an adsorbed chain generally can be displaced by another one of similar or higher molecular weight unless strong attractions are involved (for instance between a polyelectrolyte and a colloid of opposite charge18 ). In agreement with this, the presence of A 8-75 in the gradient (condition iii) induced a decrease of the amount of A*8-75 bound on porin particles. The rate of such an exchange between labeled and unlabeled amphipols appeared very slow since 40% only of bound A*8-75 was replaced by A 8-75 during a 6-h sedimentation (i.e., after migration through a volume of solution containing an amount of free polymer about 35-fold higher than the amount of bound A*8-75 and 6-fold the total amount of A*8-75 used to form the complex). Such a removal, however, was not observed on b6 f particles (Table 1). There are therefore, in this respect, IMP-related differences that will deserve further investigation. (17) Pefferkorn, E.; Carroy, A.; Varoqui, R. J. Polym. Sci. (Phys.) 1985, 23, 1997. (18) Ramachandran, R.; Somasundaran, P. J. Colloid Interface Sci. 1987, 120, 184.

The presence of surfactant in the gradient (condition iv) led to the nearly complete release of bound amphipols. In this case, the sedimentation is equivalent to the elution in a dissociating medium: excess of monomeric surfactant or micelles displaces the polymer from the IMP surface. This behavior is rather unusual as compared with the adsorption of polyelectrolyte onto charged colloids (generally they are not displaced by monomeric ions). In the present case, two main differences arise as compared with the usual associations between polymer and colloid: (i) Amphiphilic polymers display a strong affinity for surfactants. Soluble complexes between hydrophobically modified poly(acrylic acid) and surfactant were detected in solutions of various surfactants (ionic or neutral), including below the cmc;19,20 despite a high affinity for IMP, the presence of a large excess of free surfactant may shift the equilibrium toward the formation of amphipol/ surfactant and IMP/surfactant complexes. (ii) The cooperativity of hydrophobic association of the surfactant could explain that amphiphilic monomers can displace a polymer. Associated surfactant molecules may act as a whole, forming a supramolecular structure that can compete with the multipoint attachment of polymer chains. pH and Salt Effects. Both IMP and A 8-75 are weak polyelectrolytes. Therefore salt concentration and pH may have an influence on their association. The global charge of IMP at pH 8 was estimated from their amino acid residue composition5,6,21 by applying the Henderson-Hasselbach equation and using the mean pK of residues.22 Both porin trimer and RC bear a high negative charge (respectively -36 and -7); isoelectric points should be close to pH 5-6. In the case of cytochrome b6 f, on the contrary, the number of aspartic plus glutamic acid residues balances that of lysine plus arginine. The global charge of b6 f therefore depends mainly on the number of free C-terminus and N-terminus residues, which are not yet accurately determined.5 This charge was estimated to range from -8 to -4 per b6 f dimer. The expected repulsion, at pH 8, between any of the three IMP and the carboxylate units of amphipols must oppose hydrophobic association. Consequently, the ionic strength should have a strong influence on amphipol binding at this pH. Comparisons between high and low ionic strength were carried out with fully ionized polymers, at pH 8, which is also compatible with the preservation of the native globular structure of the proteins. Rate zonal sedimentations were carried out in sucrose gradients containing the same buffer as the layered mixture and no amphiphile (see Materials and Methods). The measured amphipol/IMP ratios (Table 2) show a clear effect of buffer concentration. The higher the salt concentration, the more amphipol molecules are bound. This result is in agreement with the expected reduction at high ionic strength of electrostatic repulsion between bound macromolecules. However, a clear correlation with the global charge of the protein was not observed: the behaviors of porin and RC appear very similar and much more sensitive to the ionic strength (increase by 1.8-fold) than that of b6 f (increase by 1.1fold) despite the higher phosphate concentration tested. The peculiar behavior of b6 f is also highlighted by the (19) Magny, B.; Iliopoulos, I.; Audebert, R.; Piculell, L.; Lindman, B., Prog. Colloid Polym. Sci. 1992, 89, 118. (20) Magny, B.; Iliopoulos, I.; Zana, R.; Audebert, R. Langmuir 1994, 10, 3180. (21) (a) de Vitry, C.; Breyton, C.; Popot, J.-L. J. Biol. Chem. 1996, 271, 10667. (b) Takahashi, Y.; Rahire, M.; Breyton, C.; Popot, J.-L.; Joliot, P.; Rochaix, J. D. EMBO J. 1996, 15, 3498. (c) Williams, J.-C.; Steiner, L. A.; Feher, G. Proteins 1986, 1, 312. (d) Inokuchi, K.; Mutoh, N.; Matsuyama, S.; Mizushima, S. Nucleic Acids Res. 1982, 10, 6957. (22) Sillero, A.; Meireles, R. Anal. Biochem. 1989, 179, 319.

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Table 2. Ratio (R) of the Number of Octyl Groups Belonging to Bound A*8-75 per IMP Particle at pH 8 in Various Buffersa b6 f

IMP tricine (mM) phosphate (mM) R

20 0 103 ( 9

RC 0 200 138 ( 5

18 0 83 ( 6

0 90 95 ( 6

0 97 112 ( 8

OmpF 0(1) 140 150 ( 15

30(2) 0 83 ( 12

6(2) 70.5 124 ( 6

50(2) 91.5 141 ( 3

a The concentrations reported in the table correspond to the buffer compositions of both the initial mixtures (IMP, A*8-75 and surfactant) and the sucrose gradients used for sedimentation of the complexes, except for: (1) gradient contained ammonium phosphate-NaOH buffer at 200 mM; (2) these values are the sum of Tricine (from the buffer) and of Na+ ions (coming from the stock solution of protein) in the initial mixture of IMP, polymer and surfactant. The gradients contained either Tricine-NaOH buffer at 20mM or only AP-NaOH buffer at 100 mM.

Table 3. Effect of pH on the Ratio of the Number of Octyl Groups Belonging to Bound A*8-75 to the Number of IMP Particlesa octyl groups/particle pH

b6 f

OmpF

8 7.3 6.5

138 ( 5 142 ( 10 159 ( 30

141 ( 3 153 ( 9 153 ( 7

a Initial mixtures (complexes, polymer excess and surfactant) were buffered with AP-NaOH buffer at 200 mM in the case of b6f and 90-95 mM in the case of OmpF; the sucrose gradients used for sedimentation analysis contained the same buffer as the layered samples.

necessity to add a very large excess of polymer (about 10×) in order to prevent precipitation, while a 2× excess is sufficient in the case of both porin and RC (Figure 2). This difference cannot be attributed to Coulombic effects and may stem from the presence of lipids in b6 f stock solutions. Both lipids and IMP are insoluble in the absence of micelles. Following the dilution/complexation procedure, lipids might compete with IMP for the formation of complexes with amphipols or could induce coprecipitations of b6 f, lipids, and polymer at low amphipol concentrations. Furthermore, a large quantity of lipids (more than 40 lipids per b6 f) is know to be bound to purified b6 f.16 Such a fluid layer of bound lipids around the protein might deeply affect its interactions with the octyl chains of amphipols. The influence of pH was investigated in a similar manner, at constant buffer concentration (ammonium phosphate-NaOH, 90 mM in the case of porin and 200 mM for b6 f stock solutions and sucrose gradients). Concentrations of other salts (