Thermodynamic Characterization of the Exchange of Detergents

BR−OTG complexes were prepared in equilibrium with lipid-free micelles of OTG prior to sequestration in APol as follows: The OTG−BR−lipid comple...
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Thermodynamic Characterization of the Exchange of Detergents and Amphipols at the Surfaces of Integral Membrane Proteins C. Tribet,*,† C. Diab‡ T. Dahmane,§ M. Zoonens,§ J.-L. Popot,§ and F. M. Winnik‡ †

Physico-chimie des Polym eres et Milieux Dispers es, UMR 7615, CNRS and Universit e Paris 6, ESPCI, 10 rue Vauquelin, F-75231 Paris, France, ‡Department of Chemistry and Faculty of Pharmacy, Universit e de Montr eal, CP 6128 Succursale Centre Ville, Montreal, QC, H3C 3J7 Canada, and §Physico-Chimie Mol eculaire des Membranes Biologiques, UMR 7099, CNRS and Universit e Paris-7, Institut de Biologie Physico-Chimique, CNRS FRC 550, 13 rue Pierre et Marie Curie, F-75005 Paris, France Received May 26, 2009. Revised Manuscript Received June 30, 2009 The aggregation of integral membrane proteins (IMPs) in aqueous media is a significant concern for mechanistic investigations and pharmaceutical applications of this important class of proteins. Complexation of IMPs with amphiphiles, either detergents or short amphiphilic polymers known as amphipols (APols), renders IMPs water-soluble. It is common knowledge that IMP-detergent complexes are labile, while IMP-APol complexes are exceptionally stable and do not dissociate even under conditions of extreme dilution. To understand the thermodynamic origin of this difference in stability and to guide the design of new APols, we have studied by isothermal titration calorimetry (ITC) the heat exchanges during two reciprocal processes, the “trapping” of detergent-solubilized IMPs in APols and the “stripping” of IMP-APol complexes by detergents, using two IMPs (the transmembrane domain of porin OmpA from Escherichia coli and bacteriorhodopsin from Halobium salinarium), two APols [an anionic polymer derived from acrylic acid (A8-35) and a cationic phosphorylcholine-based polymer (C22-43)], and two neutral detergents [n-octyl thioglucoside (OTG) and n-octyltetraethylene glycol (C8E4)]. In the presence of detergent, free APols and IMP-APol complexes form mixed particles, APol-detergent and IMP-APol-detergent, respectively, according to the regular mixing model. Diluting IMP-APol-detergent complexes below the critical micellar concentration (CMC) of the detergent triggers the dispersion of detergent molecules as monomers, a process characterized by an enthalpy of demicellization. The enthalpy of APol T detergent exchange on the hydrophobic surface of IMPs is negligibly small, an indication of the similarity of the molecular interactions of IMPs with the two types of amphiphiles. The enhanced stability against dilution of IMPAPol complexes, compared to IMP-detergent ones, originates from the difference in entropy gain achieved upon release in water of a few APol molecules (in the case of IMP-APol complexes) or several hundred detergent molecules (in the case of IMP-detergent complexes). The data account both for the stability of IMP-APols complexes in the absence of detergent and for the ease with which detergents displace APols from the surface of proteins.

Introduction Understanding biological processes at the chemical level is of fundamental importance for the development of new drugs and therapeutic strategies, the design of responsive biointerfaces, and the creation of functional biomimetic synthetic systems in all areas of materials science. Integral membrane proteins (IMPs) perform key cellular functions, such as energy transduction, transport of nutrients and drugs, cell-to-cell communication, and cellular adaptation to changes in environmental conditions. IMPs, however, are difficult to study in aqueous solutions, because of the high hydrophobicity of their exposed surface. Small, dispersive surfactants (called detergents in biology and hereafter) are generally used to provide an interface between this surface and the aqueous environment1,2 and, to some extent, to mimic the core of a lipid membrane (see, e.g., refs 3 and 4 and references cited therein). Free and IMP-bound detergent molecules are in dynamic equilibrium and exchange rapidly. When the concentration of free detergent is lowered below its critical micellar concentration (CMC), detergent-solubilized IMPs aggregate and, in general, precipitate, which is attributed to the (1) le Maire, M.; Champeil, P.; Moller, J. Biochim. Biophys. Acta 2000, 1508, 86– 111. (2) Garavito, R. M.; Ferguson-Miller, S. J. Biol. Chem. 2001, 276, 32403–32406. (3) Bond, P. J.; Sansom, M. S. P. J. Am. Chem. Soc. 2006, 128, 2697–2704. (4) Franzin, C. M.; Teriete, P.; Marassi, F. M. J. Am. Chem. Soc. 2007, 129, 8078.

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dissociation of the IMP-adsorbed detergent layer (see, e.g., refs 5-7). IMP-detergent complexes, therefore, are always handled in solutions above the CMC of the detergent. An excess of detergent, however, tends to inactivate IMPs (see, e.g., refs 2 and 8-12). IMP inactivation slows significantly if one replaces detergents with specially designed amphiphilic polymers called amphipols (APols).13,14 Each APol chain possesses a large number of hydrophobic moieties. Consequently, each chain interacts with the hydrophobic surface of IMPs15,16 via numerous points of (5) Moeller, J. V.; le Maire, M.; Andersen, J. P. Methods Enzymol. 1988, 157, 261– 270. (6) Moeller, J. V.; le Maire, M. J. Biol. Chem. 1993, 268, 18659–18672. (7) Champeil, P.; Menguy, T.; Tribet, C.; Popot, J.-L.; le Maire, M. J. Biol. Chem. 2000, 275, 18623–18637. (8) Bowie, J. U. Curr. Opin. Struct. Biol. 2001, 11, 397–402. (9) Rosenbusch, J. P. J. Struct. Biol. 2001, 136, 144–157. (10) Gohon, Y.; Popot, J.-L. Curr. Opin. Colloid Interface Sci. 2003, 8, 15–22. (11) Sanders, C. R.; Hoffmann, A. K.; Gray, D. N.; Keyes, M. H.; Ellis, C. D. ChemBioChem 2004, 5, 423–426. (12) Prive, G. G. Methods 2007, 41, 388–397 . (13) Tribet, C.; Audebert, R.; Popot, J. L. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 15047–15050. (14) Popot, J. L.; Berry, E. A.; Charvolin, D.; Creuzenet, C.; Ebel, C.; Engelman, D. M.; Fl€otenmeyer, M.; Giusti, F.; Gohon, Y.; Herve, P.; Hong, Q.; Lakey, J. H.; Leonard, K.; Shuman, H. A.; Timmins, P.; Warschawski, D. E.; Zito, F.; Zoonens, M.; Pucci, B.; Tribet, C. Cell. Mol. Life Sci. 2003, 60, 1–16. (15) Zoonens, M.; Catoire, L. J.; Giusti, F.; Popot, J. L. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 8893–8898. (16) Catoire, L. J.; Zoonens, M.; van Heijenoort, C.; Giusti, F.; Popot, J.-L.; Guittet, E. J. Magn. Reson. 2009, 197, 91–95.

Published on Web 07/14/2009

DOI: 10.1021/la9018772

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Tribet et al. Scheme 1. Chemical Structures of the Detergents and the Two Amphipols Used in This Studya

a

From ref 13 (A8-35) and ref 27 (C22-43).

attachment, which dramatically decreases its dissociation rate (see ref 17 and references cited therein). APol-trapped IMPs (see, e.g., refs 14 and 18 and references cited therein) or IMPs inserted into membranes of amphiphilic polymers19,20 retain their functions in aqueous solution, which opens the way to the use of polymerstabilized IMPs in biosensing, modulation of enzymatic activity, combinatorial drug development, separation methods, etc. The best-characterized APol so far, known as A8-35, is a random copolymer of acrylic acid, N-isopropylacrylamide, and N-n-octylacrylamide (Scheme 1).13 The solution properties of this polymer have been characterized extensively.21,22 A8-35 has been used, among other applications, to study IMPs in aqueous buffers in the absence of detergent (see, e.g., refs 14 and 23 and references cited therein), to fold IMPs to their native state (see ref 24 and references cited therein), and to immobilize IMPs onto solid supports for the purpose of ligand binding measurements25 (for reviews, see refs 11, 14, and 18). There are situations, however, in which A8-35, a weak polyacid, does not keep IMPs soluble and monodisperse in an aqueous environment. Thus, decreasing the pH of an IMP/A8-35 solution below a threshold value of ∼6.5 results in the partial neutralization of the carboxylate groups that keep the polymer soluble, which triggers the aggregation of the complexes.23 This is detrimental, for instance, for solution NMR studies of IMP structure (see ref 15). Increasing the salt concentration of the solution above (17) Zoonens, M.; Giusti, F.; Zito, F.; Popot, J. L. Biochemistry 2007, 46, 10392– 10404. (18) Breyton, C.; Pucci, B.; Popot, J.-L. In Membrane Protein Expression; Mus-Veteau, I., Ed.; The Humana Press: Totowa, NJ, in press. (19) Meier, W.; Nardin, C.; Winterhalter, M. Angew. Chem., Int. Ed. 2000, 39, 4599. (20) Stoenescu, R.; Graff, A.; Meier, W. Macromol. Biosci. 2004, 4, 930–935. (21) Gohon, Y.; Pavlov, G.; Timmins, P.; Tribet, C.; Popot, J. L.; Ebel, C. Anal. Biochem. 2004, 334, 318–334. (22) Gohon, Y.; Giusti, F.; Prata, C.; Charvolin, D.; Timmins, P.; Ebel, C.; Tribet, C.; Popot, J. L. Langmuir 2006, 22, 1281–1290. (23) Gohon, Y.; Dahmane, T.; Ruigrok, R. W. H.; Schuck, P.; Charvolin, D.; Rappaport, F.; Timmins, P.; Engelman, D. M.; Tribet, C.; Popot, J. L.; Ebel, C. Biophys. J. 2008, 94, 3523–3537. (24) Dahmane, T.; Damian, M.; Mary, S.; Popot, J. L.; Baneres, J.-L. Biochemistry 2009, 48, 6516–6521. (25) Charvolin, D.; Perez, J.-B.; Rouviere, F.; Giusti, F.; Bazzacco, P.; Abdine, A.; Rappaport, F.; Martinez, K. L.; Popot, J.-L. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 405–410.

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∼500 mM or adding calcium ions also affects the colloidal stability of IMP-A8-35 complexes.14,26,27 These limitations have prompted the development of chemically different APols26-30 (reviewed in ref 18), such as C22-43, a copolymer of Nn-octylacrylamide, N-isopropylacrylamide, and N-phosphorylcholine-N0 -ethylenedioxybis(ethyl)acrylamide.27,31 Due to the presence of the zwitterionic phosphorylcholine moieties, C22-43 keeps IMPs soluble in acidic media, at high ionic strength, and in the presence of divalent cations.31 Understanding the thermodynamics of IMP-APol assemblies is a critical step toward further applications of APols in biology and biotechnology, since it will provide guidelines for the use of the complexes and for the design of novel APols. It will yield comparative information about the properties of the detergent and the APol layer surrounding an IMP, which is of critical importance in understanding the physical basis for the biochemical stability that APols impart onto IMPs. Moreover, the system offers the opportunity to compare the behavior of detergents and amphipathic polymers as discrete self-assembled structures and upon adsorption onto hydrophobic surfaces. For the study described here, the surface of interest is the transmembrane region of the protein surface, but equivalent situations are encountered in numerous colloids of theoretical and technological importance. A key step toward this goal is to evaluate the enthalpy (ΔH) and entropy (ΔS) changes associated with the binding of APols to IMPs. The enthalpy of a ligand-substrate interaction as a function of the substrate/ligand ratio can be accessed experimentally by isothermal titration calorimetry (ITC), a technique which has found widespread applications in the study of biological systems involving protein-protein, protein-ligand, and (26) Picard, M.; Dahmane, T.; Garrigos, M.; Gauron, C.; Giusti, F.; le Maire, M.; Popot, J.-L.; Champeil, P. Biochemistry 2006, 45, 1861–1869. (27) Diab, C.; Tribet, C.; Gohon, Y.; Popot, J. L.; Winnik, F. M. Biochim. Biophys. Acta 2007, 1768, 2737–2747. (28) Prata, C.; Giusti, F.; Gohon, Y.; Pucci, B.; Popot, J.-L.; Tribet, C. Biopolymers 2001, 56, 77–84. (29) Gorzelle, B. M.; Hoffman, A. K.; Keyes, M. H.; Gray, D. N.; Ray, D. G.; Sanders, C. R. J. Am. Chem. Soc. 2002, 124, 11594–11595. (30) Sharma, K. S.; Durand, G.; Giusti, F.; Olivier, B.; Fabiano, A.-S.; Bazzacco, P.; Dahmane, T.; Ebel, C.; Popot, J.-L.; Pucci, B. Langmuir 2008, 24, 13581–13590. (31) Diab, C.; Winnik, F. M.; Tribet, C. Langmuir 2007, 23, 3025–3035.

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protein-polymer interactions32 and in the evaluation of polymerdetergent interactions.27,33-35 Analysis of the formation of an IMP-APol complex by ITC should, in principle, allow one to determine experimentally all the thermodynamic parameters required to characterize this system. The titration, however, presents experimental challenges, since direct formation of an IMPAPol complex cannot be performed by adding an APol to an IMP solution, or vice versa, given the insolubility of IMPs in water. Experimental protocols for the formation of IMP-APol complexes indeed involve the addition of APols to preformed IMPdetergent complexes, followed by removal of the detergent.14 This process, which in the following will be called protein trapping, involves several events, each having its own thermodynamic signature. Conversely, it is known that APols will desorb from the IMP surface when IMP-APol complexes are exposed to an excess of detergent micelles. This process, hereafter termed protein stripping, leads to the re-formation of IMP-detergent complexes.36 Stopped-flow F€orster resonance energy transfer (FRET) experiments conducted with a fluorescently labeled APol17 show that the displacement of APol A8-35 by detergents occurs within a few seconds, a time scale that makes the associated enthalpy changes amenable to monitoring by ITC. Thus, it is possible to determine also the thermodynamic quantities associated with the replacement of IMP-bound APols with detergent molecules. Nonetheless, from considerations of the thermodynamic parameters associated with the protein trapping or stripping processes alone, one cannot gain a detailed view of the molecular origins of the binding. It is necessary to conduct a stepwise thermodynamic reconstruction of the transfer process and to compare the energy associated with each of the associations involved, namely, detergent-APol, IMP-detergent, IMP-APol, IMP-APol-detergent, etc., under an equivalent set of conditions in terms of buffer, pH, and relative concentrations of detergents and APol. We report here an investigation of the thermodynamics of the entire transfer in the cases of two APols (A8-35 and C22-43), two detergents [n-octyltetraethylene glycol (C8E4) and n-octyl thioglucoside (OTG)], and two IMPs [bacteriorhodopsin (BR) from the plasma membrane of the archaebacterium Halobacterium salinarium and the transmembrane domain of porin OmpA from Escherichia coli (tOmpA)]. These proteins are representative of the two main IMP structural classes, namely, the β-barrel (tOmpA) and the R-helix bundle (BR). The ITC experiments were designed to reproduce as closely as possible standard IMPAPol preparation protocols and to collect information about the various steps of the exchange. ITC data are confronted with fluorescence quenching data obtained using a fluorescently labeled APol, which gives a measure of the extent of displacement of the APol upon addition of increasing concentrations of detergent. Experimental results from fluorescence measurements are found to be in good agreement with theoretical calculations of the degree of dilution of APols in mixed micelles expected from the thermodynamic analysis.

Experimental Section Materials. Water was deionized wih a Millipore Milli-Q water purification system. For ITC titrations, tetraethylene glycol (32) Ball, V.; Winterhalter, M.; Schwinte, P.; Lavalle, P.; Voegel, J.-C.; Schaaf, P. J. Phys. Chem. B 1992, 106, 2357–2364. (33) De, M.; You, C. C.; Srivastava, S.; Rotello, V. M. J. Am. Chem. Soc. 2007, 129, 10747–10753. (34) Heerklotz, H.; Tsamaloukas, A.; Kita-Tokarczyk, K.; Strunz, P.; Gutberlet, T. J. Am. Chem. Soc. 2004, 126, 16544–16552. (35) Matulis, D.; Rouzina, I.; Bloomfield, V. A. J. Am. Chem. Soc. 2002, 124, 7331–7342. (36) Tribet, C.; Audebert, R.; Popot, J. L. Langmuir 1997, 13, 5570–5576.

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Scheme 2. Exchange of APol (dark blue coil) for Detergent (light blue and red objects) at the Surface of IMPs (purple cylinders), and Vice Versa, in the Diverse Titration Experimentsa

a (A) For “trapping” with APols, aliquots of a solution of detergentsolubilized IMP are injected into a large volume of an APol solution, the final concentration of detergent dropping well below its CMC. (B) “Stripping” of IMP-APol complexes from their APols by injecting them into a large excess of detergent above its CMC. (C) For “mixing”, a mixed surfactant layer around the protein is formed by gradually supplementing a solution of IMP-APol complexes with detergent, the concentration of the latter in the titration cell remaining below its CMC.

n-octyl ether (C8E4) was purchased from Bachem, and n-octyl thioglucoside (OTG) and n-dodecyl maltoside (DDM) were obtained from Sigma-Aldrich. The detergents used for protein solubilization and purification (C8E4 and OTG) were purchased from Anatrace (>99% pure grade). Amphipols A8-35 (batch FGH20) and C22-43 were prepared as reported previously (refs 22 and 31, respectively). Their structure is shown in Scheme 2. tOmpA was overexpressed as inclusion bodies in E. coli, purified, and refolded as described previously.37 All experiments with tOmpA were performed in a pH 8.0 Tris/NaCl buffer containing 20 mM Tris-HCl and 100 mM NaCl. Experiments with BR were conducted in a pH 7.0 NaH2PO4/Na2HPO4 buffer containing 20 mM phosphate and 100 mM NaCl. BR was solubilized from the plasma membrane of H. salinarium (strain S9) and purified in OTG as described previously.27 IMPs were obtained as detergent complexes (tOmpA-C8E4 in Tris buffer and BR-OTG in phosphate buffer). BR-OTG complexes were prepared in equilibrium with lipid-free micelles of OTG prior to sequestration in APol as follows: The OTG-BR-lipid complexes resulting from the solubilization of purple membranes27 were purified by ultracentrifugation (4-5 h at 55000 rpm, Beckman TLS 55 rotor) in sucrose gradients (5 to 20%, w/w) containing lipid-free OTG micelles (15 mM OTG) in phosphate buffer; the band of BR was collected and dialyzed against lipid-free OTG micelles in phosphate buffer. IMP concentrations were determined from the absorbance at 554 nm [ε=1.60 L g-1 cm-1 (see ref 23)] and 280 nm [ε=2.2  103 L g-1 cm-1 (see ref 15)] for BR and tOmpA, respectively, using a UV-visible Agilent 8453 diode array spectrometer. Synthesis of the Fluorescently Labeled APol. A8-35-NBD was obtained by coupling the fluorescent dye 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole to a sample of modified A8-35 to which ∼1.5 mol % ethylene diamine was linked by an amide bond to (37) Pautsch, A.; Vogt, J.; Model, K.; Siebold, C.; Schulz, G. E. Proteins: Struct., Funct., Genet. 1999, 34, 167–172.

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Article acrylate units, as previously described.17 The batch of A8-35NBD used in this study (batch FAPol-2) comprised approximately 0.57 mol % NBD per mole of polymer. The extinction coefficient of the polymer in water (ε340) was 0.215 L g-1 cm-1 (∼2.175 L mol-1 cm-1). The acronym A8-35NBD used in the text refers to a mixture of A8-35 and A8-35-NBD (9/1, w/w) prepared as described here. Preformed solutions in water of each polymer were mixed in the appropriate ratio; the mixed solution was lyophilized, and the powder (A8-35NBD) was dissolved in the appropriate buffer (see below).

Solutions for ITC Analyses. Preparation of APol Solutions. We prepared stock APol solutions (2-20 g/L) by dissolving freeze-dried samples in deionized water under gentle stirring for at least 2 h at room temperature. APol solutions were supplemented with concentrated Tris buffer (pH 8.0) to achieve final concentrations of 20 mM Tris and 100 mM NaCl or with NaH2PO4/ Na2HPO4 buffer (pH 7.0) to achieve final concentrations of 20 mM phosphate and 100 mM NaCl. The resulting APol solutions were dialyzed against the same buffer as the solutions of IMP-detergent complexes used for ITC analyses to ensure that the ion concentration is the same in the two solutions Preparation of IMP-APol Complexes. tOmpA-APol complexes were prepared as described in ref 15. Briefly, a 10% (w/w) solution of A8-35NBD or C22-43 in water was added to an tOmpA/C8E4 solution (2 mL, 2 g/L) in Tris buffer to yield a 1/4 (w/w) protein/APol ratio. After a 15 min incubation, SM2 BioBeads were added to the solution in a 1/10 (w/w) C8E4/bead ratio. The mixture was kept for 2 h at room temperature. tOmpA/APol solutions were then dialyzed against Tris buffer. The same procedure was used to prepare BR-APol complexes, starting from a BR/OTG solution in phosphate buffer and using either A8-35 or C22-43. In the case of BR, incubation and dialysis (overnight) were conducted at 4 °C in the dark. BR/APol solutions were dialyzed against phosphate buffer. Isothermal Titration Calorimetry. Calorimetric measurements were performed with a Microcal VP-ITC instrument having a cell volume of 1.43 mL and fitted with a 300 μL syringe whose contents were continuously stirred (300 rpm) during the titration. All measurements were performed at 25 °C. The reference cell was filled with the buffer used in the experiment. Data were analyzed using Microcal ORIGIN. The experimental heat evolved (Qi) resulting from the ith solution injection was obtained by integration of the raw data signal. The integrated molar enthalpy change per injection (Δhi, in calories per mole) was obtained by dividing Qi by the number of moles of injectant added, ni, resulting in enthalpograms, which are plots of Δhi as a function of surfactant or IMP concentration in the calorimeter sample cell.

Trapping Experiments (Scheme 2A). Experiments with tOmpA. A solution of APol (A8-35 or C22-43, at 1.0 or 5.0 g/L)

in Tris buffer was placed in the sample cell. Aliquots (8 μL) of a solution of tOmpA-C8E4 complexes in Tris buffer (1.0 or 2.0 g/L tOmpA and 10 mM unbound C8E4) were injected into the sample cell at intervals of 300 s. Control experiments involved injecting C8E4 (10 mM) (i) into Tris buffer and (ii) into APol solutions (A8-35 or C22-43, at 1.0 and 5.0 g/L) in Tris buffer. Experiments with BR. A solution of the polymer (A8-35 or C22-43, at 1.0 or 5.0 g/L) in phosphate buffer was placed in the sample cell. Aliquots (8 μL) of a BR/OTG solution in phosphate buffer (0.5 or 1.0 g/L BR, 15 mM OTG) were injected into the sample cell at intervals of 300 s. Control experiments involved injecting OTG (i) into phosphate buffer and (ii) into solutions of APols (A8-35 or C22-43, at 1.0 and 5.0 g/L) in phosphate buffer. At the end of the titration, a UV-visible spectrum of the sample was recorded to check on the preservation of the absorption band of the holoprotein at 554 nm (native BR).

Mixing and Stripping Experiments (Scheme 2B,C). Partial Exchange (mixing, Scheme 2C). A tOmpA/APol solution (tOmpA/A8-35NBD or tOmpA/C22-43, tOmpA concentration 12626 DOI: 10.1021/la9018772

Tribet et al. of 0.5 or 0.25 g/L, tOmpA/polymer ratio of 4 g/g) in Tris buffer was placed in the sample cell. Aliquots (2 μL) of a solution of C8E4 (100 mM) in Tris buffer were injected into the sample cell at intervals of 300 s. Control experiments involving the titration of a solution of C8E4 (100 mM) in Tris buffer into Tris buffer and into APol solutions in Tris buffer (A8-35 and C22-43, concentration of 2 or 1 g/L) were performed under identical conditions. Extensive Exchange (stripping, Scheme 2B). A solution of C8E4 (20 mM) in Tris buffer was placed in the sample cell. Aliquots (8 μL) of a tOmpA/A8-35 solution (tOmpA at 1 or 2 g/L, A8-35 at 4 or 8 g/L) or a tOmpA/C22-43 solution (tOmpA at 1 or 2 g/L, C22-43 at 4 or 8 g/L) were injected into the sample cell at intervals of 300 s. Control experiments were performed by injection into a solution of C8E4 (20 mM) in Tris buffer (i), (ii) A8-35 (4 or 8 g/L) in Tris buffer, or (iii) C22-43 (4 or 8 g/L) in Tris buffer. Steady-State Fluorescence Measurements. Measurements were taken on a PTI spectrofluorimeter (Photon Technology International, London, ON). The excitation wavelength was 280 nm for tOmpA tryptophan residues. Emission spectra recorded between 290 and 550 nm displayed a maximum of emission of tryptophan at 330 nm. The excitation and emission slits were set at 5 and 1 nm, respectively. Samples (0.025 or 0.001 g/L tOmpA with A8-35-NBD at a 4/1 g/g APol/protein ratio in Tris buffer) were placed in 10 mm quartz cuvettes (Hellma) and stirred. Aliquots of detergent stock solutions (0.8-20 μL of ∼100 g/L dodecylmaltoside or C12E8) were added to IMP-APol solutions and the mixtures incubated for a few minutes prior to measurement. Since A8-35-NBD absorbs slightly at 280 nm (see ref 17), emission spectra were corrected for the contribution of NBD fluorescence by subtracting the emission spectrum of a control sample containing the same total concentration of A8-35-NBD in buffer. The reference emission intensity of tryptophan at 330 nm was taken as the emission of tryptophan in the solution with the maximum [detergent]/(CMC + [APol]) ratio reached experimentally (∼6.5), which was considered to provide almost full dissociation of tOmpA-APol complexes (no quenching). At the concentrations used, the effect of NBD absorbance (inner filter) was negligible. The F€ orster distance between donor (tryptophan) and acceptor (NBD) molecules, R0, has been estimated previously to be 22.5 A˚, assuming a spectral overlap of 4.910-15 M-1 cm3, a quantum yield of 0.143, an orientation factor of 2/3, and a refractive index of the solution of 1.33.17 Hence, only protein-bound APols bringing the NBD into the immediate vicinity of tOmpA are expected to contribute to significant quenching.

Results Upon dissolution in water, APols self-assemble into micellelike particles with a hydrodynamic radius of ∼4 nm.22,27 In some cases, however, APol solutions also contain small amounts of aggregates21,22 (Figure 1B and similar results, not shown, at lower pH). For studies of IMP-APol complexes, especially investigations by ITC, it is important to avoid phenomena other than the well-defined self-assembling of APols (micellization-like), which occurs concurrently with assembly onto IMPs. Contributions originating from aggregation phenomena (or from the dissociation of polymer aggregates) not only make the analysis of data more difficult but also are irrelevant to biochemical applications. Subjecting APol solutions to ultracentrifugation effectively removed the trace amounts of aggregates. The hydrodynamic radius and size distribution of APols A8-35 and C22-43 in aqueous buffers at pH >7, measured by dynamic light scattering (DLS), are similar (Figure 1A), and in both cases, the polydispersity is low. A complete characterization of the micelles of A8-35 has been reported previously.22 An important advantage of C22-43, as compared to A8-35, is that it does not aggregate in solutions that have low pH values. In contrast, in solutions at Langmuir 2009, 25(21), 12623–12634

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Figure 1. Size distributions of APol in solutions as obtained by dynamic light scattering. Intensity-weighted distribution of hydrodynamic radii in solutions (2.5 g/L) of APols A8-35 and C22-43 in 0.1 M NaCl at various pH values. (A) In 20 mM boric acid-NaOH buffer (pH 9.0), the solutions were ultracentrifuged for 10 min at 200000g in the MLA 100.2 rotor of a Beckman ultracentrifuge prior to the measurements. (B) In 20 mM NaH2PO4/Na2HPO4 buffer (pH 6.8), the solutions were simply filtered through 0.22 μm Millex syringe filters prior to the measurements.

Figure 2. Thermograms of the calorimetric titration of IMP-detergent complexes into APols. (A) tOmpA-C8E4 into 1 g/L APol C22-43 solutions [titrant, 10 mM C8E4 or 1.2 g/L tOmpA in 10 mM C8E4; buffer, 20 mM Tris-HCl (pH 8.0) and 100 mM NaCl]. (B) BR-OTG into 5 g/L C22-43 [titrant, 15 mM OTG in 20 mM NaH2PO4/Na2HPO4 (pH 7.0) and 100 mM NaCl in the presence or absence of 0.9 g/L BR].

pH 5/1; cf. ref 36) using volumes such that the final detergent concentration in the sample cell was well below the CMC (Scheme 2A). At the end of the titration, we recorded the UV-visible absorption spectrum of the solution recovered in the sample cell to ascertain (i) the absence of large aggregates, which are readily detected by drifts of the baseline in the short wavelength range (not shown), and (ii) the preservation of the native state of BR, recognized by the presence of the holoprotein absorption band at 554 nm (see ref 23 and references cited therein). Representative thermograms are shown in Figure 2, along with traces recorded upon dilution of pure detergent micelles into APols. Heat is evolved upon addition to the APol of either IMP/detergent or detergent solutions, resulting in a sharp signal which rapidly returns to the baseline. Langmuir 2009, 25(21), 12623–12634

The narrow peak width (∼1 min) and the stability of the baseline are good indications that equilibrium is reached rapidly. The addition of a given IMP-detergent complex to a given APol solution can be either exo- or endothermic, depending on the concentration of APol. The same behavior was observed upon addition of detergent to APol solutions in the absence of IMP.31 It reflects the equilibration of the detergent between free monomers (exothermic demicellization) and mixed APol/detergent particles (endothermic formation of complexes, whose ΔH is opposite to but nearly equal in magnitude to the ΔH of demicellization). The experimental heat exchange values, normalized to the molar amount of detergent injected, yield the molar enthalpies of mixing, which are plotted in Figure 3. Note that the concentration and the molar amount of detergent used in this normalization do not include the amount of detergent originating from the injected IMP-detergent complex. This is the simplest way to compare samples identical in composition for both the buffer and the unbound detergent, but with different concentrations of IMP. Under these conditions, any variation with respect to the reference enthalpy curve obtained in the absence of IMP can be ascribed to the contribution of IMP-detergent complexes to the heat evolved. The range of detergent concentrations swept during one titration was typically from 0 to ∼CMC/10. Changes in the DOI: 10.1021/la9018772

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Figure 3. Enthalpies of dilution of protein-detergent complexes into APol solutions (Scheme 2A). (A and B) tOmpA-C8E4 in 20 mM Tris-HCl (pH 8.0) and 100 mM NaCl, into (A) 5 g/L A8-35 and (B) 1 g/L C22-43 in the same buffer. (C) BR-OTG in 20 mM NaH2PO4/Na2HPO4 (pH 7.0) and 100 mM NaCl, into 1 g/L C22-43 in the same buffer.

concentration of detergent in the cell will affect the composition of the IMP-APol-detergent complexes at equilibrium: the ternary complexes are almost devoid of detergent when the concentration of unbound detergent is low (first injections), while at the end of the titration, they comprise a mixed APol-detergent belt. For a given IMP-APol-detergent system, the molar enthalpy remained constant within ∼10% throughout the titration. This result suggests that IMP trapping by the APol and the concomitant 12628 DOI: 10.1021/la9018772

Tribet et al.

release of detergent take place over the entire titration range. In contrast, the molar enthalpy of mixing depends significantly on the nature of both the IMP-detergent complexes and the APol. From the titration data, we extracted the enthalpic signature, Qtrap, for trapping of the protein by the APol. This term refers to the formation of IMP-APol complexes at the expense of IMPdetergent ones, in the presence of unbound APol/detergent mixed micelles and detergent monomers under the CMC (Scheme 2A). Qtrap is the difference between the amounts of heat associated with dilution into APol solutions of either IMP-detergent complexes, which are in equilibrium with unbound detergent at a concentration Cd slightly above the CMC, or pure detergent aliquots at concentration Cd. The Qtrap values normalized to the molar amount of protein, NIMP, are listed in Table 1. They correspond to the average of the second to fifth injections. Under these experimental conditions, which entail extensive dilution (to more than ∼25-fold below the CMC of the detergent), nearly complete desorption of the detergent from the transmembrane domain of the proteins is expected to occur (see below and Figure 4). A number of significant trends emerge within the uncertainties on the measurements. Thus, absolute Qtrap values almost double when the protein concentration increases by a factor of 2. Once normalized to the amount of protein, Qtrap values range from -40 to ca. -50 kcal/mol of IMP for the tOmpA-C8E4 pair and from ca. -240 to -340 kcal/mol of IMP for the BR-OTG pair, whatever the nature of the APol (A8-35 or C22-43) and its concentration (1 or 5 g/L) (Table 1). A priori, five processes can contribute to the exchange of heat in the trapping process: (1) demicellization of free detergent micelles, (2) dissociation of the detergent from the IMP transmembrane region and release of detergent monomers in the bulk, (3) formation of mixed micelles between detergent molecules not bound to IMPs and APols, (4) association of APols with the protein, and (5) dilution of APol particles, IMPs, and detergent micelles and monomers. In the following, to facilitate the discussion, the corresponding amounts of heat exchange are provisionally denoted Q1-Q5. We have shown previously that the dilution of APols and detergent makes a negligible contribution to the overall heat exchange,31 a conclusion that can legitimately be extended to IMPs. Q5, therefore, is neglected in the following derivations. Accordingly, to determine the enthalpy [ΔHdfA = (Q2 + Q4)/ NIMP] corresponding to the exchange of the detergent for APol at the surface of the protein, we must subtract from Qtrap the contributions of the formation of mixed micelles between the detergent released by IMPs and the unbound APol present in the solution. The amounts of heat corresponding to demicellization of free detergent micelles (Q1) and of the formation of APol/ detergent mixed micelles in the bulk with detergents that were not bound to IMP have already been subtracted by definition of Qtrap (vide supra). The amounts of heat and enthalpy corresponding to demicellization and formation of APol/detergent mixed micelles upon mixing detergent aliquots with an APol solution were determined from eq 1:31 Q1 þ Q3 ¼ Ntot;d ΔHexp ≈ ðNd -Nmic;d ÞΔHmic;d þ

Np Nd βRT Nd þ Np

ð1Þ

where ΔHmic,d is the enthalpy of micellization of the detergent, Ntot,d and Nmic,d are the molar amounts of total detergent injected and of detergent injected in micellar form, respectively, Nd is the molar amount of detergent molecules that associate with APols, Langmuir 2009, 25(21), 12623–12634

Tribet et al.

Article

Table 1. Heats Evolved upon Injection of IMP/Detergent Solutions into APol Solutions (normalized by the molar amount of IMP, NIMP)a IMP in titration cell

APol in cell (concentration)

Qtrap/NIMP (kcal/mol)

tOmpA (1.2 g/L)

C22-43 (1 g/L)

-46b

tOmpA (1.2 g/L)

C22-43 (5 g/L)

-40

tOmpA (0.6 g/L)

A8-35 (1 g/L)

tOmpA (1.2 g/L)

A8-35 (5 g/L)

b

ΔHdfA (kcal/mol) -63b -111

error in ΔHc (kcal/mol) 13

b

26

-87d

-115

19

-52

-161

19

-270

b

-276

b

24

BR (0.9 g/L)

C22-43 (1 g/L)

BR (0.9 g/L)

C22-43 (5 g/L)

-335

-370

29

BR (1 g/L)

A8-35 (1 g/L)

-307

-320

23

BR (1 g/L) A8-35 (5 g/L) -237 -290 30 a As seen in Figure 3, the enthalpies of mixing can vary by up to ∼50-100 cal/mol of detergent over the course of a titration. In detergent-into-APol titrations, this drift has been ascribed to the nonideality of mixing and to the gradual increase in the fraction of detergent in mixed APol/detergent micelles.31 In most cases, the drift of the amount of heat evolved during titrations in the presence of IMP was similar to that recorded for the same APoldetergent combination in the absence of IMPs. Qtrap/NIMP, the difference between the two measurements normalized to the amount of IMP injected, tends therefore to vary relatively little during the course of a titration. Measurements performed with solutions containing low protein concentrations (0.5 or 0.6 g/L) are, however, more sensitive to error, because of the smaller difference between the heat evolved in the presence and absence of IMPs. There was accordingly a slight difference in the drifts observed with and without proteins in dilute samples, which translates into a small change of Qtrap as a function of IMP concentration. This uncertainty was set, somewhat arbitrarily, to 50 cal/mol of detergent to estimate the impact of experimental errors. Parameters used in equations: β = 1.1, and for C8E4, ΔHmic = 4000 cal/mol and CMC = 8.2 mM; for OTG, ΔHmic = 1300 cal/mol and CMC = 9 mM. Amounts of IMP-bound detergent in tOmpA-C8E4 and BR-OTG complexes: 86 and 100 mol/mol, respectively. b Within an uncertainty of 10% (tOmpA) or 5% (BR), the Qtrap/NIMP ratio did not vary with a 2-fold dilution of the protein in the aliquots delivered to the titrating cell. For data without superscript, Qtrap/NIMP varies by ca. 30% depending on the concentration of the IMP in the titration cell. c Variation of ΔHdfA corresponding to an estimate of experimental uncertainty of (50 cal/mol of detergent in the measurements and a typical 10% uncertainty in the amount of IMP-bound detergent. d Because of the low concentration of protein as compared to other samples, the value of the heat evolved suffers from a larger uncertainty.

Figure 4. Molar fraction Xd of detergent in A8-35/detergent mixed micelles, as calculated from eq 2. (A) Mixtures of A8-35 and C8E4, using the following parameters: CMC = 8.2 mM and β=0 (solid lines) or β=1 (dashed lines); shaded areas delimit the compositions used in ITC experiments for titration of tOmpA-A8-35NBD complexes by C8E4, aiming at partial (light blue) or almost complete (light green) dissociation of A8-35 (see the text). (B) Comparison of the dequenching experimentally observed in tOmpA/A8-35-NBD/detergent mixtures (symbols) as compared to Xd values predicted from eq 2 (lines). The percentage of dequenching is the ratio of the fluorescence intensity at 330 nm to the maximum fluorescence reached in excess detergent [i.e., at [detergent]/(CMC+[APol])>6.5]: (2) C12E8 and 0.1 g/L A8-35-NBD (0.025 g/L protein) and (b) DDM and 0.004 g/L A8-35-NBD (0.001 g/L protein). Lines are calculated values of Xd in solutions of A8-35-NBD at the same concentrations as for fluorescence experiments. APol concentrations were either similar to (0.1 g/L) or much lower than (0.004 g/L) the CMC of the detergents (0.09 mM for C12E8, i.e., 0.048 g/L; 0.17 mM for DDM, i.e., ∼0.09 g/L). Experimental data were collected during the course of the FRET experiments described in ref 17.

Np is the total molar amount of APol alkyl groups, and β is an index of nonideal mixing that was found to vary between 0 and 2 depending on the detergent-APol pair (see below). The validity of eq 1 has been established previously on the basis of titrations of detergent micelles in buffer and in APol solutions (in the absence of IMP),31 and Nd was derived from the model developed in the same article. Also in the same reference,31 the concentration of free detergent in equilibrium with mixed APol/detergent micelles is written as ½detergentfree ¼ Xd  CMC  exp½ βð1 -Xd Þ2  Langmuir 2009, 25(21), 12623–12634

ð2Þ

where Xd is the molar fraction of detergent. When Xd=Nd/ (Np+Nd) , 1 (low detergent molar fraction), we have Nd ≈ Ntot;d

½APol CMC  exp β þ ½APol

ð3Þ

where [APol] is the molar concentration of n-octyl groups linked to the APol and CMC is the critical micellar concentration of the detergent. The values calculated using a β of 1.1 are listed in Table 2 together with the experimental “blank” values of the DOI: 10.1021/la9018772

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Tribet et al. Table 2. Enthalpies of Mixing of Detergent into APol Solutionsa OTG (15 mM)

C8E4 (10 mM)

ΔHexp (cal/mol)

ΔHcalc (eq 1)

ΔHexp (cal/mol)

ΔHcalc (eq 1)

none

-530

-520

-760

-720

A8-35 (1 g/L)

-380

-384

-410

-395

A8-35 (5 g/L)

-110

+11

+555

+555

C22-43 (1 g/L)

-500

-441

-570

-528

APol

C22-43 (5 g/L) not determined -183 +80 +105 a The experimental error is ca. 20 cal/mol. Parameters used in eq 1: β = 1.1; for C8E4, ΔHmic = 4000 cal/mol and CMC = 8.2 mM; for OTG, ΔHmic = 1300 cal/mol and CMC = 9 mM.

enthalpy of dilution of detergent in APol solutions in the absence of IMP. The good agreement between calculated and experimental values vouches for the validity of eqs 1 and 2 under the conditions of the titrations. Finally, the term Qtrap - NIMPΔHdfA can be calculated as the amount of heat released upon association onto APol/detergent micelles of the detergents that were initially bound to IMPs and have detached themselves from the protein. This term corresponds to the difference in the heat evolved by the association of all detergent to the APol in titrations of IMP and in the blank experiment. The blank experiment in this case is a titration into APol of a protein-free detergent solution that contains an amount of detergent identical to the amount of unbound detergent (micelles and monomers) in IMP/detergent solutions. Accordingly, the value of Qtrap - NIMPΔHdfA can be expressed from eq 1 as a function of the molar amounts of polymer-bound detergent NdIMP and Ndblank (eq 4). The NdIMP and Ndblank values differ as a result of the release of detergent from IMP-detergent complexes. This difference may be estimated from eq 3, knowing that each molecule of tOmpA binds 86 ( 12 C8E4 molecules,17 and assuming, by analogy with measurements with Triton X-100 and C12E8,6 that each BR binds ∼100 OTG molecules, which enables one to determine the total amount of detergent, Ntot,dIMP. Qtrap - NIMP ΔHdfA ≈ ðNd IMP-Nd blank ÞΔHmic;d ! Nd IMP Nd blank þ Np βRT Np þ Nd IMP Np þ Nd blank

ð4Þ

The ΔHdfA values obtained using eq 4 are listed in Table 1. The exchange of detergent for APol at the surface of the protein is exothermic in all cases. For any given protein, the molar heat released is on the same order of magnitude (∼120 kcal/mol of tOmpA and ∼320 kcal/mol of BR) whatever the experimental conditions. The uncertainty of the values is rather large [ca. ( 30 kcal/mol (Table 1)], but consistent with the differences in ΔHdfA between different entries in Table 1. ΔHdfA is different from, but on the same order of magnitude as, the enthalpies of detergent demicellization; indeed, after normalization to the molar amount of IMP-bound detergents, we obtain ca. -1400 cal/mol of C8E4 and ca. -3400 cal/mol of OTG. On the basis of the data collected in this first set of titrations, one cannot however determine the relative contributions to ΔHdfA of (i) the binding of APols to the protein and (ii) the dissociation of detergent from IMP-detergent complexes. Additional information was obtained from examining the reverse process, namely, titrating IMP-APol complexes with detergents, as described in the following section. Dissociation of IMP-Bound Amphipol in the Presence of Detergent (protein stripping). Heat exchanges associated with 12630 DOI: 10.1021/la9018772

the displacement of IMP-bound APol by detergents were studied by injecting APol-trapped IMPs into detergent solutions, or vice versa (Scheme 2B,C). Two extreme situations were explored, namely, conditions of excess detergent, for which full removal of APols from the transmembrane region of IMP is expected17 (Scheme 2B), and conditions of low detergent/APol concentration ratios, where only partial exchange will take place (Scheme 2C). To guide us in choosing concentrations of the IMP-APol complex and detergent adequate to model each situation, we assumed that the composition of the APol-detergent layer is likely to follow trends similar to those of the composition of mixed micelles in bulk. Accordingly, conditions for stripping of APol would correspond to a negligible equilibrium fraction of APol in mixed micelles, whereas partial detachment of APol from IMP is expected if unbound micelles contain a balanced amount of detergent and APol. Using eq 2, we calculated, with parameters corresponding to the A8-35/C8E4 system, the fraction, Xd, of detergent in mixed APol/detergent micelles as a function of sample concentration over an extended range. The calculated Xd values are plotted in Figure 4 as a function of C8E4 concentration normalized by CMC + [APol], where [APol] is the molar concentration of APol octyl chains. This normalization was selected so that curves corresponding to low and high detergent concentrations can be plotted on the same graph, in a manner independent of the concentration of polymer (when β = 0 and the detergent concentration is low, eq 3 shows that the parameter [detergent]/(CMC + [APol]) equals the molar ratio of APolbound detergents to the octyl groups of APol, irrespective of the CMC and APol concentrations). In this representation, the formation of mixed micelles with a dominant molar fraction of APol takes place for [detergent]/(CMC + [APol]) values of 80 mol %) occurs for [detergent]/(CMC + [APol]) . 1. The colored areas in Figure 4A correspond to the experimental ITC conditions for low (blue) and high (green) detergent ratios. The composition of ternary complexes among tOmpA, A8-35, and either C12E8 or dodecyl maltoside (DDM) as a function of detergent and APol concentrations has been estimated previously on the basis of fluorescence experiments using an NBD-labeled APol (A8-35-NBD).17 The NBD-tryptophan F€orster distance is ∼2.2 nm.17 When tOmpA is trapped by A8-35-NBD, NBD acts as an energy acceptor for its tryptophan residues and quenches their emission, while the fluorescence of NBD is enhanced. Addition of detergent to a tOmpA-A8-35-NBD solution triggers an increase in the tryptophan emission (decrease in FRET efficiency), due to desorption of A8-35-NBD from the protein. Data collected in the course of this earlier study have been analyzed in Figure 4 within the framework of the present formalism. Assuming, as a first Langmuir 2009, 25(21), 12623–12634

Tribet et al.

Article Table 3. Specific Enthalpies (normalized by the mass of APol) of Mixing of APol or IMP-APol Complexes (stripping) in 20 mM C8E4a ΔHmix (cal/g of APol) APol BR

tOmpA

a

Figure 5. Specific enthalpies of dilution of APol (A8-35NBD) or tOmpA-A8-35NBD complexes into 20 mM Tris-HCl (pH 8.0) and 100 mM NaCl, in the presence or absence of C8E4 (20 mM).

approximation, that the fluorescence of the tryptophan residues varies proportionally to the amount of A8-35-NBD bound to the protein, then the ratio I/(Imax - Imin), where I is the fluorescence intensity at λem =330 nm, Imin is the intensity in the absence of detergent (maximal quenching by A8-35-NBD), and Imax is the intensity in excess detergent [[detergent]/(CMC + APol) > 6.5], gives a measure of the composition of the tOmpA-detergentAPol ternary complexes. In Figure 4B, we have plotted as a function of [detergent]/(CMC + [APol]) the experimental values of I/(Imax - Imin) and the molar fractions of detergent in mixed micelles, calculated according to eq 2. The remarkable agreement between calculated and experimental values, at least for Xd < 80 mol %, gives strong support to the validity of our approach. The discrepancies noted for Xd values of >80 mol % can be attributed to the inadequacy of the model in predicting accurately the micelle composition in this detergent concentration domain, as reported previously.31 On the basis of this analysis, we selected ITC conditions corresponding to concentration ranges within the blue- and green-shaded areas of Figure 4A, which correspond to partial and nearly complete displacement by detergent of proteinbound APol, respectively. To study the APol-for-detergent exchange at the IMP surface under conditions of large detergent excess, aliquots of tOmpA trapped in 2 g/L A8-35NBD [A8-35NBD refers to a mixture of A835 and A8-35-NBD (see Experimental Section)] were injected into a micellar solution of C8E4 (20 mM). The weight ratio of tOmpAbound APol is estimated to be ∼1.3 g/g,17 while the overall ratio of A8-35NBD to tOmpA is 4 g/g. Thus, we estimate that, in these samples, ∼1/3 of the polymer is bound to the protein. This will be released as free APol following injection into the detergent solution. Dilutions of A8-35NBD and tOmpA/A35-85NBD solutions into detergent-free buffer were performed as control experiments, as well as a titration of A8-35NBD into micellar C8E4 (20 mM). The corresponding enthalpograms, normalized by the mass of A8-35NBD injected into the cell, are presented in Figure 5. For all titrations, the heat evolved per injection is constant over the entire experiment. In the case of the tOmpA-A8-35NBD titration, this result indicates that the enthalpy of stripping does not depend on the concentration of either APol or protein in the titration cell. We subtracted from the raw data the heat evolved upon injection of buffer into C8E4 to take into account the Langmuir 2009, 25(21), 12623–12634

IMP-APol

A8-35 (2.5 g/L)

+1.8

+2.05

C22-43 (2.5 g/L)

+0.73

-0.74

C22-43 (4.4 g/L)

+1.06

+1.00

C22-43 (8.8 g/L)

+1.11

+1.16

A8-35NBD (4 g/L)

+2.38

+2.00

A8-35NBD (8 g/L) +2.41 +2.06 Total APol/IMP = 4/1 g/g for tOmpA and 2.8/1 g/g for BR.

contribution of the slight demicellization of C8E4 upon dilution during the course of the titration. The corrected enthalpies of addition of APol, ΔHmix(APol), and of the IMP-APol complex, ΔHmix(IMP-APol), to excess C8E4 are listed in Table 3. The enthalpy of exchange, ΔHAfd = ΔHmix(IMP/APol) ΔHmix(APol), is ∼0 ( 0.1 cal/g of polymer for tOmpA-C22-43 complexes and approximately -0.35 cal/g of polymer (i.e., approximately -28 kcal/mol of protein) for tOmpA-A8-35 complexes. A high estimate of the uncertainty on each ΔHmix value is ∼10%, due to errors in concentration and heat determinations, hence errors of ∼0.15 and 0.4 cal/g of polymer on ΔHAfd for the C22-43 and A8-35 systems, respectively. Consequently, we cannot ascribe any enthalpic signature for the exchange of C22-43 for C8E4 on the surface of tOmpA. In the case of A8-35NBD, ΔHAfd may be barely in excess of the experimental uncertainty. If there is any (exothermic) contribution at all, it is less than 30 kcal/ mol of IMP, which is much smaller than the enthalpy of demicellization of C8E4 (ΔHdemic ∼ -4000 cal/mol of C8E4, vide supra, corresponds here to -328 kcal/mol for dissociation of the ∼82 molecules contained in a micelle; the latter number is similar to the estimate of 86 ( 12 molecules of C8E4 bound per tOmpA17). In conclusion, if any exchange of heat at all is associated with the displacement of either APol by C8E4, it is much smaller than the heat of C8E4 self-association or the heat of transfer of a detergent monomer into APol micelles. Titrations conducted with the BR-APol systems turned out not to be as reproducible as those described above in the case of tOmpA-APol systems, the heats of mixing varying by more than 50% for different batches of BR. BR tends to retain tightly bound endogenous lipids after detergent solubilization, and it carries them into BR-APol complexes.23 The fluctuations in the ITC data may reflect rather complex contributions of lipid interactions with the other amphiphiles. Nonetheless, the enthalpy values listed in Table 3 lead one to conclude that the exchange energetics involved are similar in the BR-APol and tOmpA-APol systems. Dilution of IMP-APol Complexes into Detergent-Free Buffer. To examine the stability of IMP-APol complexes upon dilution, we monitored by ITC the dilution of IMP-APol complexes into buffer in the absence of detergent. In control experiments, protein-free APol solutions were diluted into buffer. The heat evolved upon dilution of either tOmpA-APol complexes or pure APols up to 180-fold into surfactant-free buffer (i.e., down to 0.0056 g/L tOmpA and 0.022 g/L total APol) is very low [ca. 300 cal/g of polymer (Figure 5)]. This suggests that APols neither disaggregate into individual molecules nor dissociate from the surface of IMPs upon dilution, or that the enthalpy changes associated with these processes are very small. The stability of DOI: 10.1021/la9018772

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Figure 6. Calorimetric titration of the mixing of C8E4 and tOmpA-A8-35NBD solutions in 20 mM Tris-HCl (pH 8.0) and 100 mM NaCl. (A) Thermograms of the dilution of the tOmpA-A8-35NBD complexes (solid black line, 1 g/L tOmpA, 4 g/L A8-35NBD) and A8-35NBD (4 g/L, open red circles) into 20 mM C8E4. (B) Titration of 100 mM C8E4 into (4) the tOmpA-A8-35NBD complex (0.5 g/L tOmpA, 2 g/L A8-35NBD), (b) 2 g/L A8-35NBD, or (9) buffer.

IMP-APol complexes at extreme dilutions is consistent with FRET measurements indicating that tOmpA-A8-35-NBD complexes (2.5 g/L of tOmpA and 10 g/L of polymer) preserve their integrity for as long as 24 h upon 1000-fold dilution in surfactantfree buffer.17 Similarly, FRET measurements using a pair of fluorescent APols (unpublished data) indicate that A8-35 forms micelles in buffer solutions at concentrations as low as 0.002 g/L, a value well below the lowest concentration employed in ITC titration (0.01 g/L). These observations confirm that neglecting the contribution of any eventual APol demicellization processes is justified in analyzing these experiments. Injecting Detergent into an IMP-Bound APol Layer. Finally, we conducted titrations aimed at probing the interactions between detergent and IMP-APol complexes in the concentration domain dominated by IMP-APol mixed micelles [0 < Xd< 60 mol % (green area in Figure 4A)]. A micellar solution of C8E4 (100 mM) was injected into solutions of either A8-35NBD or the tOmpA-A8-35NBD complex, the overall APol concentration of which was the same (Scheme 2C). Thermograms are presented in Figure 6A. In both traces, the heat signal recorded upon injection is sharp with a rapid return to baseline values, indicating that equilibration in the cell occurs within ∼1 min. The heat evolved upon injection is identical for the two systems throughout the titrations, which covered a C8E4 concentration range from 0 to 9-10 mM and Xd values from 0 to ∼60 mol %. The uncertainty in the measurements was estimated to be on the order of 0.1 kcal/ mol of detergent, a value well below the enthalpy of demicellization. As a control, we monitored the dilution of C8E4 into buffer. This results in demicellization of C8E4, a process characterized by a thermodynamic profile very different from the profiles observed upon titration in APol, as seen in Figure 6B. The enthalpies of C8E4 dilution in buffer and in APol differ by 0.6-1.7 kcal/mol (Figure 6B). The lack of influence of the presence or absence of IMPs points to a negligibly small enthalpic contribution of any interaction of the detergent with the protein. Thus, in this concentration domain also, the enthalpy of exchange of APol for detergent at the surface of IMPs solely reflects detergentAPol interactions, and there is no indication that the detergent interacts differently with free and protein-bound APol.

Discussion The heat evolved during the exchange of detergent for APol (or vice versa) around the hydrophobic surface of IMPs reflects 12632 DOI: 10.1021/la9018772

structural changes affecting the coverage of the IMP hydrophobic belt by alkyl groups as well as the energy associated with the selfassembly of the released amphiphile in water. The ITC measurements led to experimental values of the enthalpy of the exchange of IMP-bound detergent for APol, ΔHdfA, and of the enthalpy of the reverse process, the replacement of IMP-bound APol by detergent, ΔHAfd. One should compare these enthalpies with the enthalpies of micellization, in the absence of IMP, to obtain the order of magnitude of the energy involved in the formation of these self-assembled structures. Noting that ΔHAfd is significantly smaller than the enthalpy of demicellization of the detergent, one can conclude that the protein transmembrane region is masked similarly from contacts with water whether it is trapped by APols or by detergents. In addition, the estimated enthalpies of formation of ternary complexes (IMP-APol-detergent) indicate that the partitioning of the detergent into APol does not depend on the presence of a protein. Comparison of the Enthalpy Changes during the Processes of Protein Trapping (ΔHdfA) and Protein Stripping (ΔHAfd). The experimentally recorded large difference in magnitude between the enthalpy, ΔHdfA, ascribed to the sequestration of IMPs in APols [dilution of IMP-detergent complexes into an excess of APol, approximately -100 to -400 kcal/mol of IMP (Table 1)] and the enthalpy, ΔHAfd ∼ 0, related to the exchange of APol for detergent (dilution of IMP-APol complexes into excess detergent) raises several questions. The ΔHdfA term contains the energy involved in the replacement of detergent with APols as well as the energy associated with the demicellization of the IMP-bound detergent released as monomer in bulk water. The enthalpy of the reverse process (ΔHAfd) reflects only the replacement of the APol by detergent on the IMP: since the concentration in bulk water of the APol released remains well above its CAC, there is no demicellization term associated with ΔHAfd. Assuming that the processes taking place on the surface of IMPs are essentially reversible and should have opposite enthalpic contributions in the trapping and stripping experiments, the quantity ΔHdfA - ΔHAfd should be identical to the enthalpy of demicellization of the detergents used. Experimentally, this is not the case. An exact calculation is hard to do in the case of BR, because there is no precise estimate of the amount of OTG it binds. However, in the case of tOmpA, SANS contrast-matching experiments have yielded a reasonably precise estimate of 86 (12 molecules of C8E4 bound per tOmpA monomer.17 Langmuir 2009, 25(21), 12623–12634

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The corresponding demicellization enthalpy would be ca. -330 to 400 kcal/mol of tOmpA. Experimentally, ΔHdfA - ΔHAfd ≈ -120 kcal/mol of tOmpA (Tables 1 and 3). The difference between the experimental enthalpy (this work) and the estimated value implies that the steps in the d f A and A f d exchange processes are not symmetrical. During the exchange of APols for detergents (above the CAC of APol), the transmembrane region of the IMP is permanently protected from contact with water, because the rate of detergent adorption on the IMP is expected to be much faster (on a time scale of microseconds; see ref 38) than the APol stripping process (on a time scale of tens of milliseconds; see ref 17). What follows the dilution of IMP-detergent complexes into APols is not as clear-cut. Upon dilution below the CMC, at least part of the bound detergent is likely to dissociate much more rapidly (probably on a time scale of microseconds again) than the APol associates (under our salt conditions, APolAPol exchange at the surface of tOmpA takes place on a time scale of minutes17). The interval of time between desorption of the detergent and adsorption of the APol may give the protein an opportunity to form small aggregates, which may not redisperse sufficiently fast on the time scale of ITC measurements or may not redisperse at all. We have shown previously that the order in which APol addition and detergent removal are performed can affect the outcome of protein trapping experiments: for the same final IMP and APol concentrations, trapping measured as retention of the protein in the supernatant upon ultracentrifugation, which removes all but very small aggregates, is more efficient if the polymer is added before diluting the detergent under its CMC than if IMP-detergent complexes are diluted into the APol solution.36 The d f A ITC experiments presented here mimic the latter trapping conditions. It is therefore possible that, in the d f A ITC experiments, the formation of IMP oligomers competes kinetically with that of IMP-APol monomeric complexes. The final objects, thus, would be different from the monodisperse complexes used as a starting material in the A f d experiments. In particular, they could feature some protein-protein interactions, formed at the detriment of protein-APol interactions. Experimental parameters specific to the ITC technique may also affect the results. Our derivations are based on the hypothesis that equilibrium is reached at the end of the titrations. It has been shown experimentally, by FRET timeresolved measurements, that such is the case for the stripping process, which takes place within the time scale of sequential ITC injections.17 On the other hand, we have no direct evidence that the dfA exchange occurs as quickly. Consequently, we cannot be certain that all of the final IMP-APol contacts had formed within the time frame of the ITC titration experiments. Origin of the Thermodynamic Stability of IMP-Amphipol Complexes. Previous studies on the structure and function of IMP-APol complexes have demonstrated that they have many similarities with those of IMP-detergent complexes and that it is possible to convert IMP-detergent complexes into IMP-APol complexes, and vice versa, without IMP denaturation or aggregation. This study demonstrates that the IMP-amphiphile binding enthalpy is very similar, if not identical, for the two types of surfactants. IMP-APol complexes exhibit a remarkable stability upon dilution in buffer. This resistance to dilution seemingly contrasts with the ease of APol T detergent exchange. A priori, the persistence of the IMP-APol association upon dilution may be ascribed to either kinetic and/or thermodynamic effects. Exchange kinetics are fast, as shown by ITC and FRET experiments, (38) Frindi, M.; Michels, B.; Zana, R. J. Phys. Chem. 1992, 96, 6095–6102.

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and will be considered as nonlimiting in the following discussion, which, therefore, focuses on thermodynamics. Since the enthalpic contributions to adsorption of detergent and APol onto IMP are indistinguishable, the higher stability of IMP-APol complexes must be a consequence of entropic contributions. The main entropic difference can be assumed to be translational entropy, rather than a difference of solvation, given that the hydrophobic groups carried by the two types of amphiphiles are the same (n-octyl). The desorption of APols from an IMP-APol complex, which would lead to IMP aggregation, would release, in the cases studied here, at most three to five (macro)molecules per protein, which, given the very low CAC, would, in most cases, remain associated with each other. In contrast, aggregation upon dilution below the detergent CMC of IMP-detergent complexes releases ∼100 detergent monomers per protein. The translational entropy gain under these conditions (TΔS ∼ RT ln Nagg ∼ 300 kcal/mol of protein) is therefore an efficient driving force toward detergent release and IMP aggregation. The stability of IMP-APol association can therefore be attributed to the low CAC of APols, a concentration which is usually not reached by simple dilution, and to the low entropy gain achieved upon disassembly of the complexes. The thermodynamic stability of the complexes shown in this work may not be the only factor involved; one may envisage also that the complexes gain additional protection because of the slower kinetics of detachment of adsorbed macromolecules, compared to “small” detergents. However, kinetic barriers are not sufficient, per se, for protecting the complexes under all the conditions commonly encountered during the preparation and purification of IMPs. In solutions with ionic strength on the order of 100 mM, the exchanges of APol for detergent and even the APol-APol exchanges are rapid (on the order of minutes17) and thermodynamic stability is accordingly important for preserving IMP/APol complexes. The entropic effects described above can also explain the ease of exchange between detergent and APol on the surface of IMPs. In this case, one should compare the stability of IMP complexes from the point of view of the exchange of micelles of APols for micelles of detergents. For the small IMP studied here, both IMP-APol and IMP-detergent complexes feature protein/micelle stoichiometries on the order of ∼1/1. Hence, in terms of translational entropy, APol or detergent micelles pay a similar energy cost to form complexes. The absence of either enthalpic or entropic bias in favor of one surfactant or the other is consistent with the experimental observation that detergent micelles and APol micelles compete about equally well for the surface of the protein. From a practical point of view, this means that, in ternary mixtures, the composition of the surfactant layer surrounding the protein will roughly reflect the overall ratio of APol to micellar detergent.

Conclusion Investigations by fluorescence and ITC have shown that the enthalpy of exchange of small surfactants (detergents) for APols on the surface of IMPs is negligibly small, compared to the enthalpy of micellization of the detergents. Since the enthalpy is sensitive to the self-assembly process, the fact that the APoldetergent exchange is isoenthalpic implies that the organization of the two types of amphiphiles around the protein is quite similar, irrespective of their different chemical structures. In the absence of an enthalpic driving force that would favor one type of complex over the other, the entropy of binding dominates the equilibrium. Assuming that translational entropy plays the major role, the success of IMP trapping upon dilution below the CMC of the DOI: 10.1021/la9018772

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detergent of a ternary IMP-APol-detergent mixture can be attributed primarily to the entropic gain resulting from the release of tens of detergent molecules in the solvent, while at the same time, only a few APol chains bind to the protein. Conversely, stripping of APol-bound IMP and the resulting reassembly of IMP-detergent particles can be readily achieved, as long as the detergent concentration in solution is above the CMC. Data from both ITC and FRET experiments concur to indicate that detergent and APols bind to IMP in proportions approximately equal to their ratio in mixed micelles. Other aspects of the ITC data reported here have not yet been exploited to their full extent. For example, we noted that when the titrations were conducted by dilution of IMP-detergent complexes below the detergent CMC into an APol solution, the enthalpies of exchange differed from zero. This difference suggests that additional processes take place during the exchange, most likely some level of protein aggregation. This result may explain the experimental observation that the preparation of IMP-APol complexes is more efficient if ternary complexes (APol-detergent-IMP) are assembled in the presence of detergent micelles, followed by removal of excess detergent, rather than by carrying out simultaneously the addition of APol and the removal of detergent.36 Thermodynamic studies ought to contribute to an improved understanding of such empirical rules and eventually lead to a more rational use of APols for the handling and study of IMPs in aqueous solutions. Acknowledgment. Particular thanks are due to F. Giusti (UMR 7099) for the synthesis of A8-35 and A8-35-NBD, to Y. Gohon (UMR 7099) for his participation in early experiments, and to both of them for discussions and comments on the manuscript. J.-L.P. thanks the Center of Self-Assembly and Colloid Sciences (FQRST) for travel support. Work in F.M.W.’s laboratory was supported by a grant from the Natural Sciences and Engineering Research Council of Canada. Work in J.-L.P.’s laboratory was supported by the CNRS, by University Paris-7,

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and by a grant from the EU (Specific Targeted Research Project LSHG-CT-2005-513770 IMPS Innovative tools for membrane protein structural proteomics). Work in C.T.’s laboratory was supported by ANR-BLAN-07-0278.

Glossary Abbreviations Ntot,d: total molar amount of detergent injected into the ITC cell. Nmic,d: molar amount of detergent injected in the form of micelles, i.e., a molar amount of detergent that contributes to the heat ascribed to demicellization upon dilution. Nd: molar amount of detergent in the cell (after injection) that associates with APol at equilibrium. Np: molar amount of octyl groups attached to APols in the ITC cell. NIMP: molar amount of membrane protein in the cell. ΔHdfA: molar enthalpy of dissociation of IMP-bound detergent (released as monomers) and their replacement by IMP-bound APol (normalized to 1 mol of IMP). ΔHAfd: molar enthalpy of dissociation of IMP-bound APol (released as micelles) and their replacement by IMP-bound detergents (normalized to 1 mol of IMP). ΔHmic,d: molar enthalpy of micellization of the detergent. CMC: critical micellar concentration of the detergent (N.B.: The critical self-association concentration of APols, CAC, is assumed here to be negligibly low as compared to experimental concentrations). β: index of nonideality of mixing APols with detergent under the form of micelles (β=0 corresponds to zero enthalpy of mixing in the pseudophase model). Xd: molar fraction of detergent in APol-detergent mixed assemblies at equilibrium (N.B.: For the polymer, the molar amount corresponds to the amount of octyl side groups).

Langmuir 2009, 25(21), 12623–12634