Binding of water-soluble, globular proteins to anionic model membranes

Journal of Molecular Structure 924–926 (2009) 274–284

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Binding of water-soluble, globular proteins to anionic model membranes Francisco Torrens a,*, Gloria Castellano b, Agustín Campos c,1, Concepción Abad d a

Institut Universitari de Ciència Molecular, Universitat de València, Edifici d’Instituts de Paterna, P.O. Box 22085, E-46071 València, Spain Instituto Universitario de Medio Ambiente y Ciencias Marinas, Universidad Católica de Valencia San Vicente Mártir, Guillem de Castro-94, E-46003 València, Spain Institut Universitari de Ciència dels Materials, Universitat de València, Dr. Moliner-50, E-46100 Burjassot (València), Spain d Departament de Bioquímica i Biologia Molecular, Universitat de València, Dr. Moliner-50, E-46100 Burjassot (València), Spain b c

a r t i c l e

i n f o

Article history: Received 3 October 2008 Received in revised form 9 December 2008 Accepted 12 December 2008 Available online 25 December 2008 Keywords: Protein–lipid interaction Binding isotherm Partition coefficient Ionic strength Salt effect

a b s t r a c t The role of electrostatics is studied in the adsorption of proteins to negatively charged (phosphatidylcholine/phosphatidylglycerol, PC/PG) and neutral (PC) small unilamellar vesicles (SUVs). For model proteins the interaction is monitored vs. pH at low ionic strength. The adsorption behaviour of lysozyme, myoglobin and albumin (isoelectronic point, pI 5–11) is investigated in SUVs, along with changes of the fluorescence emission spectra of the charged proteins, via their adsorption on SUVs. Significant adsorption of the proteins to negatively charged SUVs is found only at pH values, where the number of positive charge moieties exceeds the number of negative charge moieties on the protein, by at least 3 e.u. The fluorescence emission of positively charged proteins increases, after adsorption on negatively charged SUVs. With increasing protein to phospholipid ratio, the increase in the fluorescence emission levels off and reaches a plateau. Neutralization of the SUV charge is the controlling factor in their adsorption. The plateau level depends on the type of protein and pH of the incubation medium. The pH dependency can be ascribed to the mean positive charge of the protein. The effective charge of all proteins is calculated from the charge differences between empty and protein-coated SUVs. Ó 2008 Elsevier B.V. All rights reserved.

1. Introduction The interaction between proteins and small unilamellar vesicles (SUVs) can be mediated by electrostatic and/or hydrophobic forces, or by covalent bonds. Intrinsic membrane proteins can be embedded within the SUV bilayer, via hydrophobic interactions between the hydrophobic parts of the protein and the hydrocarbon chains of the phospholipids. The association of water-soluble proteins with vesicles is thought to be primarily dependent on the overall electrostatic, Coulombic attraction between the membrane- and protein-associated charges [1,2]. However, others reported that the association is mainly dependent on hydrophobic interactions; the charge characteristics of the protein play only a minor role [3–5]. The relative significance of the interactions is difficult to assess. The physicochemical properties of the proteins (size, shape and charge), presence of hydrophobic patches on the surface of the protein, conformation of the protein (formation of amphipathic a-helices), vesicle characteristics and incubation conditions can affect the binding characteristics [6,7]. Several studies showed that the association of positively charged proteins with negatively charged membranes is enhanced, compared to the association with neutral membranes [8–10]. Low-molecular-weight synthetic polypeptides * Corresponding author. Tel.: +34 963544431; fax: +34 963543274. E-mail address: [email protected] (F. Torrens). 1 A.C. deceased on May 14, 2008. 0022-2860/$ - see front matter Ó 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.molstruc.2008.12.040

were employed to unravel the role of the sign, number of charges, charge distribution and overall hydrophobicity of the polypeptide, in polypeptide–membrane interactions [11–15]. The affinity of positively charged mono and divalent peptides for cardiolipin (CL) vesicles is determined by both hydrophobic and electrostatic interactions, while the main determinant for zwitterionic membranes is the overall hydrophobicity of the peptide [13]. The distribution of the charged moieties in divalent, positively charged peptides has little influence on the affinity of the peptide for CL. The membrane association of polypeptide melittin was evaluated [16–18]. Although the binding of melittin to SUVs is because of hydrophobic effects, electrostatic interactions increase the concentration of peptide adjacent to the membrane and enhance the binding. The electrostatic effects were analyzed with the Gouy– Chapman theory. The net molecular charge, calculated for melittin, was substantially lower than that predicted on the basis of its molecular structure. For proteins that contain several positively and negatively charged groups, either homogeneously distributed or regionally enriched, the situation is even more complex. Little detailed information is available about the role of the protein charge, in the association with SUVs. The role of protein charge was investigated on association with preformed, negatively charged vesicles composed of phosphatidylcholine (PC)/phosphatidylglycerol (PG), for a set of model proteins [19]. The ionic strength was kept low to promote electrostatic interactions. Protein adsorption, electrophoretic mobility and vesicle size were

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measured under identical conditions vs. pH. Mean protein charge was assessed by acid/base titration. Lysozyme hydrolyzed carbohydrate linkages in bacterial cell walls. It induced fusion on negatively charged phospholipid vesicles, and electrostatic interactions were important [20]. It interacted with PC/phosphatidic acid (PA) and PC vesicles; the release and fusion of PC/PA vesicles were observed [21]. It was established a mechanism with a relationship between aggregation, leakage and fusion of vesicles induced by lysozyme interaction. It was studied the aggregation, fusion and aqueous content release from PC/PA, PC and PC/dioleoylphosphatidylethanolamine (DOPE)/cholesterol (Chol) vesicles, induced by lysozyme derivatives, and their effect on the lytic activity [22]. The lysozyme-induced fusion of phosphatidylserine (PS) vesicles was pH dependent, and the maximum occurred at pH 5 [23]. A penetration of lysozyme into the phospholipid membrane was observed, using energy transfer measurements and monolayer techniques. Lysozyme-induced fusion resulted from the penetration of lysozyme into the hydrophobic core of the bilayer, occurring at acidic pH. Lysozyme bound to negatively charged phospholipid vesicles over a broad pH range [24]. The amount of binding is regulated by electrostatic forces. In dependence on pH, the bound protein induced a strong destabilization of the negatively charged phospholipid bilayers. The destabilization was connected with the reduced water content in the phospholipid head-group region, because of aggregation, exposure of hydrophobic patches and total reorganization of the vesicles. It was reported the salt-induced liquid–liquid phase separation of lysozyme–sodium dodecylsulfate (SDS) complexes, in solution [25]. It was studied the interactions of lysozyme with SDS and Triton X (TX)-100 micelles, and reversed micelles of sodium bis(2ethylhexyl)sulphosuccinate (Aerosol-OT, AOT) [26]. Lysozyme inserted into monoglyceride monolayers and was able to induce a lamellar gel to coagel phase transition, in monoglyceride bilayers [27]. The insertion of b-lactoglobulin (b-LG) depended on the lipid composition of the monolayer. It was promoted when the acyl chains of the negatively charged amphiphile were shorter than those of the monoglyceride. The binding parameters were similar for the interaction of b-LG and lysozyme with monoglyceride bilayers. A large exothermic binding enthalpy was observed, which depended on the nature of the monoglycerides. The effect of lysozyme stability on protein–monoglyceride interactions was studied [28]. It was analyzed the thermotropic phase behaviour of a 1-[2H35]-stearoyl-rac-glycerol ([2H35]-MSG)/dicetylphosphate (DCP) mixture and its interaction with lysozyme, by 2H- and 31Pnuclear magnetic resonance (NMR) [29]. It was examined the interaction of surfactants cetyltrimethylammonium bromide (CTAB) and SDS with lysozyme, by isothermal titration microcalorimetry vs. protein concentration, pH and temperature [30]. The protein-induced aggregation of the surfactants was observed. The bovine serum albumin (BSA) molecule consists of a chain of 582 amino-acid residues, forming a single polypeptide, which contains three homologous a-helix domains (I–III), which are divided into nine loops by 17 disulphide bonds [31,32]. Its primary structure is unusual in possessing a single sulfhydryl group (Cys-34). Every domain can be divided into 10 helical segments. The molecule is not uniformly charged within the primary structure. At pH 7.0 net charges of 10.0, 8.0 and 0.0 (total 18.0 e.u.) were calculated for domains I, II and III, respectively [33]. In earlier publications, it was studied the binding of vinyl polymers to anionic model membranes [34], interaction of polyelectrolytes with oppositely charged micelles [35] and association of melittin with neutral phospholipid vesicles [36], by spectrofluorimetry and liquid chromatography. In the present report lysozyme, myoglobin and BSA were used as model proteins because they are water-soluble, globular proteins with known three-dimensional structures, covering a wide range of pH of isoelectronic point (pI,

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5–11). Some of their properties relevant for this study are calculated. Monodomain lysozyme and myoglobin are similar in size and dimension, but they differ in other physicochemical properties, which will help to reduce the effect of molecular size on their associations with vesicles [37]. However, tridomain BSA is greater. Lysozyme is characterized by a higher content of charged amino acids, while BSA is characterized by their relatively lower content. The surface hydrophobicity of the proteins is relatively low in the native state; e.g., the retention time of lysozyme and myoglobin on hydrophobic-interaction chromatography columns is short, compared to that of BSA [38,39], which often has been used in protein–lipid interaction studies. Lysozyme and myoglobin show no major conformational changes between pHs 4 and 10 [40]. Varying the pH within this range, the effect of charge on protein–SUV interactions can be studied, for a protein without major conformational changes. Charge differences between empty SUVs and SUVs with lysozyme, myoglobin or BSA adsorbed on their surface were calculated from spectrofluorimetric data, using the Gouy–Chapman theory. A satisfactory correlation was found between the calculated effective charge, defined as the number of PG molecules neutralized by one adsorbed protein molecule, and the mean protein charge, determined on the basis of acid/base titration of lysozyme and myoglobin.

2. Materials and methods 2.1. Materials Phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidylinositol (PI), cardiolipin (CL), hen egg-white lysozyme, horse heart myoglobin and bovine serum albumin (BSA) were from Sigma (St. Louis, Mo. USA). Egg-yolk phosphatidylcholine (PC) was purchased from Merck (Darmstad, Germany) and purified according to Singleton et al. [41]. Salt, buffers and reagents were of the highest purity available. 2.2. Vesicle preparation Small unilamellar vesicles (SUVs) composed of PC, PC/PG, PC/ stearic acid (SA) mixtures of various compositions, PS, PI and CL were prepared by dissolving an appropriate amount of lipid in chloroform/methanol. The solvent was evaporated under a stream of N2(g), and the lipid was dried under vacuum overnight. The 0.010 mol L1 3-(N-morpholino)-propanesulphonic acid (MOPS)– NaOH buffer pH 7.0, 0.010 mol L1 acetate buffer pH 4.0 or 0.010 mol L1 glycine buffer pH 9.0, at a given NaCl concentration in the range 0–1 mol L1, was added to the dry film and the suspension was extensively vortexed. The lipid dispersion was next sonicated for 20 min, at a temperature above the phase transition temperature of the phospholipid, by using an ultrasonic generator, with a microtip probe (Vibra cell, Sonics and Materials, Inc., Daubury, CT), at a power setting 4 and 50% duty cycle. The samples were then centrifuged for 15 min, at 35,000g, to remove probe particles and the remaining multilamellar aggregates. The lipid content in the resulting SUV preparations was determined by a phosphorus assay [42]. The integrity of SUV preparations was controlled by negative-stain electron microscopy [43]. 2.3. Fluorescence spectroscopy Steady-state fluorescence measurements were recorded using a Perkin-Elmer (Beaconsfield, UK) LS–50 fluorescence spectrophotometer, with 1.0  1.0 cm quartz cuvette. The excitation and emission bandwidths were 5 nm. The excitation wavelength was set to 280 nm for all proteins. Spectra were corrected by compari-

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son to quinine sulphate standard. In the binding experiments, the fluorescence emission spectra of the proteins in MOPS–NaOH pH 7.0, acetate pH 4.0 or glycine buffer pH 9.0 were monitored from 300 to 440 nm. Titrations were performed by addition of small aliquots of the SUV solution to the protein, at a desired concentration in 1 mL, and the shown data are representative of several independent experiments. The possible weak fluorescence contribution, because of the buffer and/or lipid solutions without proteins, was always used as baseline in all the experiments and subtracted. In lipid–protein mixtures the changes of the emission fluorescence intensity, at k = 345 nm (lysozyme, BSA) or k = 337 nm (myoglobin), Ik, were analyzed vs. Ri (lipid–protein molar ratio) and, from the fluorescence intensity increase, the fraction of bound protein a, defined by a = (IkIkfree)/(IkboundIkfree), was estimated as previously described [44–47]. The Ikbound value was extrapolated from a double-reciprocal plot. In all experiments the total protein concentration was 1 lmol L1. 3. Results and discussion 3.1. Binding and partition equilibrium models The association of protein molecules to lipid vesicles is generally described by a partition model, in which one considers the membrane as a separate lipid phase, in which the protein can dissolve. The partition model allows the calculation of a partition coefficient Kr for the protein, between the lipid and aqueous phases, defined as the ratio of the protein activity in the lipid phase aLp to that in the aqueous phase aAp , when secondary effects make the system to deviate from the ideal behaviour:

Kr ¼

aLp cLP cLp ¼ aAp cLP cAp

ð1Þ

where cLp and cAp are the concentrations of protein in the lipid and aqueous phases, respectively, and cLp and cAp the activity coefficients in each specific phase, attributed to electrostatic repulsions among the positive protein molecules in each phase. When the lipid volume is negligible with regard to the solvent volume, the following expression that relates cLp =cAp with experimental data can be derived:

cLP ða=Ri Þ ¼ A ð1  aÞ½PT mL cP

ð2Þ

L is the lipid partial molar volume (0.785 L mol1 [48]), and where m (1  a)[P]T the aqueous free protein concentration. Since the protein is considered having access only from the vesicle outside, a/Ri is corrected by the fraction of lipid in the outer leaflet b, i.e., a=Ri ¼ ða=Ri Þ=b, denoting the moles of adsorbed protein per mole of accessible lipid. For SUVs where ca. 2/3 of the lipids stay in the outer shell a value of b = 0.65 can be used. The following expression for describing a real partition equilibrium is obtained by substituting Equation (2) into (1):

L C ða=Ri Þ Krm ¼ ¼ ð1  aÞ½PT c c

ð3Þ

where the activity coefficient c ¼ cLp =cAp reflects possible unideal protein–protein interactions. Parameter C is proportional to the L , which is a measure of the free energy partition coefficient C ¼ K r m of the protein–lipid interactions and independent of protein concentration:

 0;A  DG  0;L DG P P C ¼ cntmL exp RT

of the protein in the aqueous and lipid phases, respectively. The va 0;A for cationic ion–water interactions is given by [49]: lue of DG P

   0;A DG NA ðzþP Þ2 e2 1 DGcav P 1 ¼ þ RT 2RTðRp þ 2Rw Þ4pe0 ew RT  

4N A zþP elw 2

RTðRp þ Rw Þ 4pe0

ð4Þ

where cnt is a constant that depends on the molar masses and den 0;A and DG  0;L the molar free energies sities of lipid and water, and DG P P

4NA zþP ehw 2RTðRp þ Rw Þ3 4pe0

4NA ðzþP Þ2 e2 aw 2RTðRp þ Rw Þ4 4pe0

ð5Þ

The free energy of solvation is composed of five terms. The first one refers to the Born charging contribution: the free-energy change resulting from the transfer of ions from vacuum to a structureless continuum medium, the water solvent with relative permittivity ew. The cavitation term involves the work of forming a cavity in the solvent, the work of splitting up the extracted solvent molecules and separating them to infinity, the work of orientating the solvent molecules around the protein and the work of condensing the solvent molecules, not used in the solvation of the ion. The three other terms refer to the ion–dipole, ion–quadrupole and ion–induced dipole interactions, respectively. All the parameters included in Eq. (5) are known, except Rp (radius of the protein ion) and zþ p , the latter being the actual protein-ion charge in solution, which can be different from the physical charge because of partial screening. The NA is the Avogadro number, e = 1.6021892  1019 C the proton charge, R = 8.3143 J mol1 K1 the gas constant, T = 293 K the temperature, Rw = 2.8 Å the effective radius of solvation of water, eo = 8.854  1012 C2 N1 m2 the absolute permittivity of vacuum, ew = 78.5 the relative permittivity of water, lw = 1.86 D the dipole moment of water, hw = 3.9  1026 statC cm2 the quadrupole moment of water, and aw = 1.65  1040 C2 m2 J1 the deformation polarizability, which is a measure of the distortability of the water molecule along its permanent dipole axis. The cavitation energy DGcav can be calculated as:

DGcav ¼ sS þ W 0 ¼ s4pR2p  RT lnð1  V w qw Þ

ð6Þ

where s is the water–air surface tension (435 J Å2 at 298 K), S the molecular surface area [50], W0 the cavitation work for a null-surface solute, Vw the volume of a water molecule and qw the water  0;A can be obtained density. An expression similar to that for DG P  0;L , including the Born charging contribution and ion–ion for DG P interactions:

   0;L DG NA ðzþP Þ2 e2 1 NA zþP zL e2 P  1 ¼ RT 2RTRp 4pe0 eL RT4peL e0 Rp ð1 þ jRp Þ þ

NA zþP zþL e2 RT4peL e0 Rp ð1 þ jRp Þ

ð7Þ

where eL is the relative permittivity of a lipid membrane,

j¼



2e2 NA I ew e0 kT

1=2 ð8Þ

 is the inverse Debye length, zþ L and zL the positive and negative charges of the phospholipid head, and k the Boltman constant. In order to evaluate the relationship between zþ p and m (protein effective interfacial charge for the membrane-bound state of the protein, in the Gouy–Chapman approach), the cLp values were used to determine m with the expression proposed by Schwarz and Beschiaschvili [51]:

1 

ln c ¼ 2m sin h

!

þ



mbða=Ri Þ

ð9Þ

where parameter b:

b¼

20e bAL ð8ew e0 RTIÞ1=2

ð10Þ

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is essentially determined by the ionic strength I of the bulk electrolyte, and AL = 70 Å2 is the estimated area of the phospholipid head [52]. Under the present experimental conditions b = 3.10(400/I)1/2. After correction for electrostatic effects by the Gouy–Chapman theory, the binding isotherms of cationic proteins on anionic vesicles could be described by a partition model. In terms of conventional binding mechanisms, which do not take into account electrostatic effects, this would correspond to a negative cooperativity. In this study binding proteins, with various surface charge characteristics, were used to test the hypothesis that an ionic interaction facilitates protein–vesicle adsorption. 3.2. Binding interface of lysozyme into lipid bilayer The lysozyme–phospholipid interaction was studied by spectrofluorimetry. To delineate the binding interface of lysozyme–SUVs, we relied on the Trp residues as distinct reporters. The approach was facilitated by the natural occurrence of surface-exposed Trp residues in lysozyme. The Trp fluorescence is strongly influenced by the indole side chain; it has proved to be a useful tool to monitor conformational changes of proteins and protein–membrane interactions [53]. When buried in a hydrophobic environment, Trp flurescence generally shifts to a shorter (blue shift) maximal wavelength, kmax, and often exhibits an increase in maximum fluorescence intensity FImax. An opposite effect is observed in a polar environment. Lysozyme possesses six Trp residues per molecule. Three out of the six Trp residues (63, 111 and 123) are surface exposed. The fluorescence emission spectrum of lysozyme exhibits a peak at 338 nm. Such intense fluorescence and the long wavelength of maximum emission, both argue for Trp residues partially buried within apolar environment [54]. The emission spectrum was recorded in the absence and presence of phospholipid SUVs. In the presence of SUVs, lysozyme showed quenching in FImax and slightly shifted kmax (3 nm) to shorter wavelengths (blue shift) by addition of SUVs, compared with lysozyme alone. The results indicate that lysozyme interacts with SUVs and that Trp residues are located at the membrane interface. The quenching of fluorescence (more exposed Trp) and blue-shifted kmax, in the presence of SUVs, clearly indicated association of lysozyme with the lipid bilayer, although the indole side chains of Trp residues appear to be localized in the polar environment, at the interface. Fluorescence quenching suggests that lysozyme could account for membrane intersection. The charges on lysozyme are +12.0, +8.0 and +6.0 e.u. at pHs 4.0, 7.0 and 9.0, respectively, and decrease rapidly as the isoelectronic point at pH 10.7 is approached. The solution of lysozyme dissolved in buffers of pH 10.0 is turbid, indicating some aggregation. Lysozyme molecules aggregate reversibly at pHs above 5.5 [55–57]. Lysozyme in solution at pH 5–9 is a self-associating system of the monomer–dimer type, while at higher pHs larger polymers appear. Dimerization is dependent on the pH, protein concentration, temperature and ionic strength. At pH 5.0–5.5, protein concentration 0.15%, temperature 20 °C and ionic strength 0.150 mol L1, lysozyme is monodisperse. The dimerization reaction is related to the ionization of a single group, with an apparent pKa = 6.2, which corresponds in value to Glu-35. The formation of the dimer will protect lysozyme molecules from hydrolysis. The experimental binding curves, obtained for lysozyme, were next analyzed using the partition equilibrium model with zþ p as adjustable parameter, assuming eL = 20 and that the protein was solvated in both aqueous and lipid phases. As a first approximation a globular shape for lysozyme (Rp = 20.3 Å) was used. The cavitation energy DGcav is estimated from the cavity surface and grows roughly quadratically with protein size. This is the largest endothermic contribution to the hydration energy, which must be overcome by the Born charging interaction. The hydration energy was

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found to be exothermic, because of the large number (28) of charged residues. The adsorption of lysozyme 1 lmol L1 on mixed PC/PG SUVs, at T = 20 °C, pH 7.0 (MOPS 0.010 mol L1) and I = 0.015 mol L1 (cf. Fig. 1), shows that protein adsorption increases with the content of anionic PG in PC/PG, as expected for the electrostatic interaction between cationic lysozyme and anionic PG. The zþ p , C = Cexp and m values are calculated for lysozyme upon binding to PC/PG SUVs, at various v/v compositions. For the PC/PG mixtures, the phospholipid charge has been treated as a fitting parameter and optimized to zL = 0.00 e.u. For every plotted experimental point a pair of zþ p and C values is obtained. In the calculations a value of eL = 2.0 was used. The zþ p ¼ 4:8  5:1 e:u is lower than the electrostatic charge of lysozyme (+8.0 e.u.), at pH 7.0. This observation can be understood, if one considers that the actual zþ p charge of cationic lysozyme should be decreased, because of the presence of counterions in the electrolyte solution (which provides a given I) [58]. The obtained overall < zþ p > values slightly decrease as the anionic PG content increases. The diminution is in agreement with the experimental fact that, in mixed anionic/zwitterionic SUVs, the anionic phospholipid is asymmetrically located in the inner leaflet of the bilayer [59,60]. On increasing the anionic PG content from 0% to 40% (v/v), = strongly increases from 1.07  105 to 6.35  105 L mol1, as expected for the electrostatic interaction between cationic lysozyme and anionic PG. Despite the decrease in < zþ p >, = 1.60 e.u. remains almost constant with PG content. Notice that in some cases of anionic vesicles (zL < 0) and high surface coverage, e.g., PC/PG (95:5, v/v, zL = 0.05 e.u.), the calculation of m did not converge. As the ad-molecule is cationic, it can be expected after adsorption that zL > 0.05 e.u. Therefore, zL has been treated as a fitting parameter and increased until m convergence. The fitting value of zL = 0.00 e.u. is also calculated. The adsorption of lysozyme 1 lmol L1 on mixed PC/PG (90:10, v/v) SUVs, at T = 20 °C and pH 7.0 (MOPS 0.010 mol L1, cf. Fig. 2), shows that adsorption decreases when the ionic strength increases, as expected for the screen of the electrostatic interaction between cationic lysozyme and anionic PC/PG, because of the partial screening of the counterions in the electrolyte solution. The zþ p , C = Cexp and m values are computed for lysozyme upon binding to PC/PG

Fig. 1. Influence of vesicle charge on the adsorption of lysozyme–PC/PG at T = 20 °C, pH 7.0 and I = 0.015 mol L1.

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Fig. 2. Influence of ionic strength on the adsorption of lysozyme–PC/PG (90:10, v/v) at T = 20 °C and pH 7.0.

90:10 (v/v) SUVs, at various ionic strengths. The phospholipid charge has been treated as a fitting parameter and optimized to zL = 0.00 e.u. For higher ionic strengths < zþ p > begins to decrease as the ionic strength increases, as usually observed for polyelectrolytes [61,62], because of the partial screening of the counterions in the electrolyte solution. Again, the obtained zþ p ¼ 4:9  5:1 e:u is lower than the electrostatic charge of lysozyme (+8.0 e.u.), at pH 7.0. Once zþ p is known C and m have been estimated vs. ionic strength. On increasing the ionic strength from 0.015 to 0.105 mol L1, < zþ p > slightly increases from 4.90 to 5.10 e.u., strongly decreases from 3.42  105 to = 1.06  105 L mol1, and strongly increases from 1.62 to 2.04 e.u., as expected for the screening of the electrostatic interaction between cationic lysozyme and anionic PG. The adsorption of lysozyme 1 lmol L1 on various anionic phospholipid SUVs, at T = 20 °C, pH 7.0 (MOPS 0.010 mol L1) and I = 0.015 mol L1 (cf. Fig. 3), shows that on both monoanionic PS and PI, surface coverage is lower than that on partially dianionic CL (charge 1.24 e.u.), as expected for the electrostatic interaction between cationic lysozyme and anionic phospholipids. Notice that CL (pKa1 = 2.8, pKa2 = 7.5) is only dianionic at greater pH. The zþ p, C = Cexp and m values are calculated for lysozyme upon binding to phospholipid SUVs of various charges. On the three monoanionic PS, PI and PG SUVs, < zþ p >¼ 5:6  5:7 e:u. is somewhat greater than that on the partially dianionic CL SUVs (5.46 e.u.). On the partially dianionic CL SUVs, = = 4.63  105 L mol1 is strongly greater than those on the monoanionic phospholipid SUVs (0.6–0.8  105 L mol1). On the monoanionic PS–PI–PG SUVs, = 0.5–0.7 e.u. is rather greater than that in the partially dianionic CL SUVs (0.16 e.u.). The adsorption of lysozyme 1 lmol L1 on PC SUVs, at T = 20 °C, I = 0.015 mol L1, and pHs 4.0, 7.0 and 9.0 (buffers 0.010 mol L1, cf. Fig. 4), shows that at pHs 7.0 and 9.0 surface coverage is greater, as expected for the fact that PC is neutral. However, at pH 4.0 adsorption is lower, as expected for the electrostatic repulsion between cationic lysozyme and the PC partial cationic charge (+0.02 e.u.). The zþ p , C = Cexp and m values are computed for lysozyme upon binding to PC SUVs at various pHs. The < zþ p > slightly decreases as pH increases. The = strongly increases from

Fig. 3. Influence of negatively charged vesicles on the adsorption of lysozyme– phospholipid at T = 20 °C, pH 7.0 and I = 0.015 mol L1.

Fig. 4. Influence of pH on the adsorption of lysozyme–PC at T = 20 °C, I = 0.015 mol L1 and pHs 4.0, 7.0 and 9.0.

0.23  105 to 0.55  105 L mol1 as pH increases, because of the loss of electrostatic repulsion between cationic lysozyme and partially cationic PC. Despite the diminution in < zþ p >, stays almost constant (1.60 e.u.) with pH. The adsorption of lysozyme 1 lmol L1 on mixed PC/PG (90:10, v/v) SUVs is calculated, at T = 20 °C, I = 0.015 mol L1 and pHs 4.0, 7.0 and 9.0 (buffers 0.010 mol L1). Surface coverage increases again with increasing pH, as expected for the fact that PC/PG is almost PC (Fig. 4). At pHs 7 and 9 PC/PG shows greater coverage than PC. In fact from pH 4 to 9, the difference between PC/PG and PC increases and surface coverage is also greater. The zþ p , C = Cexp and m are computed for lysozyme upon binding to PC/PG (90:10, v/v)

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SUVs, at various pHs. Again < zþ p > slightly decreases as pH increases. The = strongly increases from 0.43  105 to 1.59  105 L mol1 as pH increases, because of the electrostatic attraction between cationic lysozyme and anionic PG. Despite the decrease in < zþ p >, remains almost constant (1.60 e.u.) with pH. It is of interest the knowledge of the molecular details of the interaction between lysozyme and membrane phospholipids, for two reasons. (1) Lysozyme provides a convenient and suitable model for studying lipid–protein interactions involving initial electrostatic binding, followed by putative penetration into the lipid bilayer. In lysozyme this interaction is accompanied by SUV aggregation, membrane fusion and permeability changes. (2) The lysozyme–lipid interaction may provide important clues to understand bactericidal action. Lysozyme, which is positively charged at pH 7.0, produces aggregation and fusion of negatively charged SUVs. The results are consistent with the ability of electrostatic charges, in protein binding to lipid SUVs, to produce the studied phenomena. The protein acts via a conformational change, produced upon binding to the lipid vesicles, involving an increase in loops and unordered structure, at the expenses of b-sheet conformation and b-turns. The forces involved in the interaction of lysozyme with lipid vesicles are not only electrostatic. In addition, membrane destabilization also plays a role in the aggregation and fusion of lipid vesicles, induced by proteins. Lysozyme binding was demonstrated to alter the structure of the lipid phase of negatively charged membranes [63]. Although the electrostatic interactions between negatively charged membranes and lysozyme are well documented and extensively discussed, within the context of specific mechanisms of lysozyme binding, some results indicate the existence of hydrophobic interactions between the protein and the lipid bilayer. Strong experimental support for a hydrophobic component, in the interaction, was provided by the finding that all bound lysozyme cannot completely dissociate from such membranes, either by increasing the ionic strength of the buffer solution, or by dilution of the bulk protein [64]. Consistent with the electrostatic nature of the interaction, the effect occurs despite the fact that the initial binding itself is rather sensitive to both ionic strength of the solution and amount of the charge in the lipid membrane. The fact that electrically neutral lipid SUVs are able to bind lysozyme evidently indicates an affinity, based on hydrophobic interactions. On the other hand, electrostatic interactions between the positive charges of lysozyme and negatively charged SUVs do also occur. Experiments on the binding of mastoparan X (MPX) to negatively charged vesicles suggested that this goes along with farther penetration into the bilayer, leading eventually to a translation across the membrane [65]. Practically, no such translocation does occur in their electrically neutral vesicle membranes. There is also a change of the effective charge number z. In the first place, it must be pointed to the fact that zþ p is always found to be clearly smaller than the true physical charge number, which should be rather above +12.0, +8.0 and +6.0 e.u. for the pHs 4.0, 7.0 and 9.0, respectively. This peculiar phenomenon has been generally observed in analogous cases, with multicharged associated proteins. It can be attributed to a fundamental defect of the Gouy–Chapman model. The latter presumes charges being smeared over the very interface, between the water and the lipid domains. However, one deals with discrete charges, which may penetrate more or less into the electrolyte and imply excluded-volume effects. Such deficiencies of the applied model result in a reduction of the apparent charge number [52]. The observed effective value of zþ p will accordingly also be subject to changes, depending on the definite structure of the associated protein, which is salt dependent.

279

3.3. Binding interface of myoglobin into lipid bilayer The charges on myoglobin are +14.0, 0.0 and 4.0 e.u. at pHs 4.0, 7.0 and 9.0, respectively, corresponding to a pI of 6.9. The experimental binding curves obtained for myoglobin were next analyzed, using the partition equilibrium model with zþ p as adjustable parameter, assuming eL = 20 and that the protein was solvated in both aqueous and lipid phases. As a first approximation a globular shape for myoglobin (Rp = 22.6 Å) was used. The adsorption of myoglobin 1 lmol L1 on mixed PC/PG SUVs, at T = 20 °C, pH 4.0 (acetate 0.010 mol L1) and I = 0.015 mol L1 (cf. Fig. 5), shows that surface coverage increases with the content of anionic PG in PC/PG, as expected for the electrostatic interaction between cationic myoblobin and anionic PG. The zþ p , C = Cexp and m are computed for myoglobin upon binding to PC/PG SUVs at various v/v compositions. For the PC/PG mixtures, the phospholipid charge has been treated as a fitting parameter and optimized to zL = 0.00 e.u. The < zþ p > slightly decreases as PG content increases. The diminution is in agreement with the fact that the anionic phospholipid is asymmetrically located in the inner leaflet of the bilayer. On increasing the anionic PG content from 0% to 40%, = strongly increases from 0.85  105 to 3.43  105 L mol1, as expected for the electrostatic interaction between cationic myoglobin and anionic PG. Despite the diminution in < zþ p >, stays almost constant (1.69) with PG content. The adsorption of myoglobin 1 lmol L1 on mixed PC/PG (90:10, v/v) SUVs is calculated, at T = 20 °C and pH 4.0 (acetate 0.010 mol L1). Surface coverage decreases as the ionic strength increases, as expected for the electrostatic interaction between cationic myoglobin and anionic PC/PG. The zþ p , C = Cexp and m are computed for myoglobin upon binding to PC/PG (90:10, v/v) SUVs, at various ionic strengths. The phospholipid charge has been treated as a fitting parameter and optimized to zL = 0.00 e.u. The < zþ p > slightly increases, = strongly decreases from 4.62  105 to 1.31  105 L mol1, and strongly increases from 1.73 to 2.22 e.u. as the ionic strength increases. The adsorption of myoglobin 1 lmol L1 on various anionic phospholipid SUVs is calculated, at T = 20 °C, pH 4.0 (acetate 0.010 mol L1) and I = 0.015 mol L1. On the three monoanionic phospholipids CL, PG and PI, surface coverage is greater than that

Fig. 5. Influence of vesicle charge on the adsorption of myoglobin–PC/PG at T = 20 °C, pH 4.0 and I = 0.015 mol L1.

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on partially monoanionic PS (charge 0.11 e.u.), as expected for the electrostatic interaction between cationic myoglobin and anionic phospholipids. Notice that PS (pKa1 = 3.0, pKa2 = 4.6, pKa3 = 9.0) is only monoanionic at greater pH. The zþ p , C = Cexp and m are computed for myoglobin upon binding to phospholipid SUVs of various charges. The phospholipid charge has been treated as a fitting parameter, and optimized to zL = 0.00 e.u. (PG, PS, CL) or 1.0 e.u. (PI). On the formally monoanionic PG, CL and PI SUVs, < zþ p > (5.5–5.7 e.u.) is slightly smaller than that on the partially monoanionic PS (5.78 e.u.). On the formally monoanionic PG–CL–PI SUVs, = = 0.4–1.1  105 L mol1 is strongly greater than that on the partially monoanionic PS (0.23  105 L mol1). Despite the variation in < zþ p >, = 1.69 e.u. remains almost constant with phospholipid charge. The adsorption of myoglobin 1 lmol L1 on PC SUVs is calculated at T = 20 °C, I = 0.015 mol L1, and pHs 4.0, 7.0 and 9.0 (buffers 0.010 mol L1. Protein adsorption decreases when the pH increases, as expected for the corresponding change of electrostatic interactions, for myoglobin passing from cationic to neutral to anionic, respectively. The zþ p , C = Cexp and m are computed for myoglobin upon binding to PC SUVs at various pH s. The < zþ p > (5.2– 5.7 e.u.) increases as the pH increases. Notice that the formal electrostatic charge of myoglobin at pH 9.0 is negative, while zþ p is calculated positive. The corresponding interpretation is the consideration of different regions of negative and non-negative charges in myoglobin, and that its adsorption on PC SUVs is located in non-negatively charged regions. The = strongly decreases from 4.62  105 to 0.32  105 L mol1, and slightly decreases as the pH increases. The adsorption of myoglobin 1 lmol L1 on mixed PC/PG (90:10, v/v) SUVs is calculated, at T = 20 °C, I = 0.015 mol L1, and pHs 4.0, 7.0 and 9.0 (buffers 0.010 mol L1). Surface coverage decreases again as the pH increases (myoglobin from cationic to anionic). In particular at pH 4 the coverage on PC/PG is lower than that on PC. The zþ p ,C = Cexp and m are computed for myoglobin upon binding to PC/PG (90:10 v/v) SUVs, at various pHs. The < zþ p > (5.6– 5.8 e.u.) increases, = strongly decreases from 0.79  105 to 0.20  105 L mol1, and is almost constant (1.69 e.u.) as the pH increases.

intrinsic protein fluorescence is the high sensitivity of Trp to its local environment. The BSA domains have a hydrophobic interior and polar exterior; almost all hydrophobic residues are located between the helices and inside the trough, whereas the great majority of polar residues are on the outer wall of the structure. The environments of Trp(s) in BSA solutions are relatively polar, and the environment of Trp-134 presents higher polarity than Trp212 [69]. The adsorption of BSA 1 lmol L1 on PC SUVs, at T = 20 °C and pH 7.0 (MOPS 0.050 mol L1, cf. Fig. 6), shows that surface coverage decrases with increasing ionic strength I. The zþ p ,C = Cexp and m are calculated for BSA upon binding to PC SUVs at various ionic strengths. For moderate ionic strengths the < zþ p > begins to decrease as the ionic strength increases, as usually observed for polyelectrolytes, because of the partial screening of the counterions in the electrolyte solution. Notice that the formal electrostatic charge of BSA at pH 7.0 is negative, while zþ p is calculated positive. As there exist three domains (I–III) in BSA with recalculated net charges of 5.0, 4.0 and 0.0 e.u., the adsorption on PC SUVs is expected from the neutral domain III. In the whole range of ionic strength < zþ p > slightly increases, = strongly decreases from 0.84  105 to 0.61  105 L mol1, and strongly increases from 2.13 to 4.91 e.u. The adsorption of BSA 1 lmol L1 on mixed PC/stearic acid (SA) (90:10, v/v) SUVs is calculated, at T = 20 °C, pH 7.0 (MOPS 0.050 mol L1) and I = 0.025 mol L1. Surface coverage on PC/SA is lower than that on PC (Fig. 6). The zþ p , C = Cexp and m are computed for BSA upon binding to PC/SA (90:10, v/v) SUVs. The mean average 5 1 values are < zþ p >¼ 6:95 e:u:, = = 0.84  10 L mol and = 2.13 e.u. We did not always observe a real plateau for the isotherm. The observations fit well with the commonly held view that hydrophobicity is the predominant driving force for the binding of anionic ligands, e.g., fatty acids (BSA is the transporter of cholesterol, fatty acids and anions via the blood), but assisted by electrostatic interactions between the phosphate group and basic amino-acid residues [70–74]. Since binding of BSA to vesicles suggests a local, not global negative domain this may occur at pH < pI.

3.4. Binding interface of bovine serum albumin into lipid bilayer The charges on BSA are +15.0, 9.0 and 33.5 e.u. at pHs 4.0, 7.0 and 9.0, respectively, corresponding to a pI of 5.5, i.e., the charges on BSA are always negative at pHs 7.0 and 9.0 whereas positive at pH 4.0. At pH 7.0 net charges of 5.0, 4.0 and 0.0 e.u. for domains I, II and III, respectively, are recalculated for the experimental total charge of 9.0 e.u. The experimental binding curves, obtained for BSA, were next analyzed using the partition equilibrium model with zþ p as adjustable parameter, assuming eL = 20 and that the protein was solvated in both aqueous and lipid phases. As a first approximation a globular shape for BSA (Rp = 31.5 Å) was used. In the present study, the quenching of the intrinsic fluorescencence of BSA was observed by selectively exciting Trp residues. The BSA contains two Trp residues (134 and 212). Measurement of quenching of BSA’s natural fluorescence is an efficient method to study its interaction with several substances. It can reveal the accessibility of quenchers to BSA’s fluorophore groups, help understand BSA’s binding mechanisms to drugs and provide clues to the nature of the binding phenomenon [66,67]. A solution of BSA excited at 290 nm emits fluorescence, attributable mainly to its Trp residues. The Trp-134 is located in subdomain IB and Trp-212 in subdomain IIA. The adherence of two subdomains with their grooves towards each other forms a domain, and three of such domains make up a BSA molecule [68]. An important feature of

Fig. 6. Influence of ionic strength on the adsorption of BSA–PC at T = 20 °C and pH 7.0.

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3.5. General discussion of the three proteins binding interfaces into lipid bilayers Table 1 specifies the mean average values of zþ p and C, used for the evaluation of the theoretical binding isotherms, and the molar free energies of the proteins in the aqueous and lipid phases. For a given binding isotherm, the m values are constant between the experimental error and strongly smaller than zþ p . An effective charge, smaller than that expected from the number of ionizable groups, is a well-known phenomenon previously described for

peptides [51,17,75–78]. Additionally, the use of the partition equilibrium model, in combination with the Gouy–Chapman formalism, provides an expression to generate theoretical association isotherms, which depend on two adjustable parameters, viz. C (calculated from the initial slope of the experimental curves), and m. The theoretical isotherms look qualitatively similar to those obtained by means of the thermodynamic approach. Notice that C values, theoretically calculated for the three proteins, are of the same order of magnitude than those derived from the fitting analysis, meaning that for each binding isotherm, the evaluated value

Table 1  0;A and in the lipid Mean average values of the actual vesicle charge in solution zL, protein charge in solution zþ p , the molar free energies of the protein in the aqueous phase DGP  0;L per mole of protein ions, the theoretical partition coefficient C and the effective interfacial charge m for the binding of protein to phospholipid vesiclesa. phase DG P System b

Lysozyme/PC Lysozyme/PC/PG 95:5b Lysozyme/PC/PG 90:10b Lysozyme/PC/PG 80:20b Lysozyme/PC/PG 60:40b Lysozyme/PC/PG 90:10c Lysozyme/PC/PG 90:10d Lysozyme/PC/PG 90:10e Lysozyme/PSb Lysozyme/CLb Lysozyme/PIb Lysozyme/PGb Lysozyme/PCf Lysozyme/PCb Lysozyme/PCg Lysozyme/PC/PG 90:10f Lysozyme/PC/PG 90:10b Lysozyme/PC/PG 90:10g Myoglobin/PCf Myoglobin/PC/PG 95:5f Myoglobin/PC/PG 90:10f Myoglobin/PC/PG 80:20f Myoglobin/PC/PG 60:40f Myoglobin/PC/PG 90:10f Myoglobin/PC/PG 90:10h Myoglobin/PC/PG 90:10i Myoglobin/PC/PG 90:10j Myoglobin/PC/PG 90:10k Myoglobin/PC/PG 90:10l Myoglobin/PGf Myoglobin/PSf Myoglobin/CLf Myoglobin/PIf Myoglobin/PCf Myoglobin/PCb Myoglobin/PCg Myoglobin/PC/PG 90:10f Myoglobin/PC/PG 90:10b Myoglobin/PC/PG 90:10g BSA/PCc BSA/PCm BSA/PCn BSA/PCo BSA/PC/SA 90:10c a b c d e f g h i j k l m n o

zL

< zþ p >

 0;A (J mol1) 104 DG P

 0;L (J mol1) 104 DG P

105 (M1)

105 (M1)



0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.00 1.24 1.00 1.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

5.09 4.93 4.89 4.82 4.77 5.03 5.11 5.10 5.66 5.46 5.64 5.71 5.35 5.21 5.20 5.25 4.98 5.02 5.55 5.95 5.62 5.46 5.30 5.24 5.53 5.29 5.43 5.75 5.48 5.62 5.78 5.51 5.67 5.24 5.55 5.73 5.56 5.56 5.81 6.95 7.07 6.97 7.22 6.95

62.27 57.83 56.73 55.08 53.85 60.55 62.82 62.47 78.66 72.63 77.96 80.11 69.62 65.58 65.36 66.58 59.19 60.33 68.40 79.78 70.11 65.81 61.56 60.04 67.76 61.36 64.99 73.90 66.52 70.11 74.77 67.32 71.65 60.04 68.21 73.29 68.72 68.66 75.58 82.20 85.30 82.57 89.12 82.20

66.07 61.86 60.82 59.25 58.09 64.44 66.60 66.27 82.37 76.79 81.70 83.75 73.05 69.21 69.00 70.16 63.15 64.23 72.15 82.97 73.77 69.68 65.64 64.20 71.53 65.46 68.90 77.38 70.37 73.77 78.20 71.12 75.23 64.20 71.96 76.79 72.45 72.39 78.97 85.94 88.93 86.34 92.78 85.94

1.07 2.71 3.42 4.88 6.35 1.53 0.96 1.06 0.73 4.63 0.84 0.56 0.23 0.53 0.55 0.43 2.03 1.59 0.85 0.09 0.60 1.43 3.43 4.62 0.96 3.55 1.71 0.29 1.31 0.60 0.23 1.05 0.44 4.62 0.88 0.32 0.79 0.80 0.20 0.84 0.51 0.95 0.61 0.84

1.07 2.71 3.42 4.88 6.35 1.53 0.96 1.06 0.73 4.63 0.84 0.56 0.23 0.53 0.55 0.43 2.03 1.59 0.85 0.09 0.60 1.43 3.43 4.62 0.96 3.55 1.71 0.29 1.31 0.60 0.23 1.05 0.44 4.62 0.88 0.32 0.79 0.80 0.20 0.84 0.51 0.95 0.61 0.84

1.60 1.60 1.62 1.60 1.60 1.64 1.78 2.04 0.54 0.16 0.50 0.66 1.61 1.60 1.60 1.60 1.60 1.59 1.67 1.70 1.69 1.68 1.69 1.73 1.69 1.71 1.82 1.92 2.22 1.69 1.69 1.67 1.69 1.73 1.70 1.69 1.69 1.71 1.69 2.13 3.03 3.67 4.91 2.13

In all calculations eL was 20.0 and it was considered that the protein is solvated in both aqueous and lipid phases. pH 7.0, I = 0.015 M, T = 20.0 °C. pH 7.0, I = 0.025 M, T = 20.0 °C. pH 7.0, I = 0.055 M, T = 20.0 °C. pH 7.0, I = 0.105 M, T = 20.0 °C. pH 4.0, I = 0.015 M, T = 20.0 °C. pH 9.0, I = 0.015 M, T = 20.0 °C. pH 4.0, I = 0.020 M, T = 20.0 °C. pH 4.0, I = 0.025 M, T = 20.0 °C. pH 4.0, I = 0.040 M, T = 20.0 °C. pH 4.0, I = 0.055 M, T = 20.0 °C. pH 4.0, I = 0.105 M, T = 20.0 °C. pH 7.0, I = 0.125 M, T = 20.0 °C. pH 7.0, I = 0.275 M, T = 20.0 °C. pH 7.0, I = 1.025 M, T = 20.0 °C.

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Table 2  0;A  0;L Mean average values of the actual protein charge in solution zþ p , the molar free energies of the protein in the aqueous phase DGP and in the lipid phase DGP per mole of protein ions, the theoretical partition coefficient C and the effective interfacial charge m for the binding of protein to phosphatidylcholine/phosphatidylglycerol (PC/PG) 90:10 (v/v) vesiclesa. Protein

I (M) 0.015

0.020

0.025

0.040

0.055

0.105

0.125

0.275

1.025

Lysozyme < zþ p >  0;A (J mol1) 104 DG P  0;L (J mol1) 104 DG P 5 10 (M1) 5 10 (M1)

4.89 56.73 60.82 3.42 3.42 1.62

– – – – – –

5.03 60.55 64.44 1.53 1.53 1.64

– – – – – –

5.11 62.82 66.60 0.96 0.96 1.78

5.10 62.47 66.27 1.06 1.06 2.04

– – – – – –

– – – – – –

– – – – – –

Myoglobin < zþ p >  0;A (J mol1) 104 DG P  0;L (J mol1) 104 DG P 105 (M1) 5 10 (M1)

5.24 60.04 64.20 4.62 4.62 1.73

5.53 67.76 71.53 0.96 0.96 1.69

5.29 61.36 65.46 3.55 3.55 1.71

5.43 64.99 68.90 1.71 1.71 1.82

5.75 73.90 77.38 0.29 0.29 1.92

5.48 66.52 70.37 1.31 1.31 2.22

– – – – – –

– – – – – –

– – – – – –

BSA < zþ p >  0;A (J mol1) 104 DG P  0;L (J mol1) 104 DG P 105 (M1) 5 10 (M1)

– – – – – –

– – – – – –

6.95 82.20 85.94 0.84 0.84 2.13

– – – – – –

– – – – – –

– – – – – –

7.07 85.30 88.93 0.51 0.51 3.03

6.97 82.57 86.34 0.95 0.95 3.67

7.22 89.12 92.78 0.61 0.61 4.91

a

In all calculations eL was 20.0 and it was considered that the protein is solvated in both aqueous and lipid phases.

of zþ p originates a theoretical value of C similar to that obtained from the initial slope. However, with the present thermodynamic approach, the determination of C and m parameters is made separately, without any extrapolation from the experimental isotherm. Notice also that both theoretically calculated and experimental are different for the various proteins, because of their wide range of pI (5–11). Table 2 summarizes the average < zþ p > values, obtained for the proteins upon binding to phospholipid SUVs at various ionic strengths. For moderate ionic strengths, < zþ p > slightly decreases as the ionic strength increases, as usually observed for polyelectrolytes, because of the partial screening of the counterions in the electrolyte solution. We obtain zþ p values lower than the electrois known, the free molar energies of the prostatic charges. Once zþ p  0;A and lipid phases DG  0;L , have been teins, between the water DG P P  0;A and estimated vs. the ionic strength and C values. Both DG P 0;L  DGP display negative values and, for moderate ionic strengths, their absolute values decrease as the ionic strength increases. At first glance, the fact reveals that the thermodynamic process of dissolving the proteins in both phases is spontaneous, as observed for  0;A  DG  0;L are positive values the three proteins. The differences DG P P and, for moderate ionic strengths, slightly increase with the ionic strength, denoting a more favourable thermodynamic process of the proteins to be dissolved in the lipid phase. Notice that for a given ionic strength, the values of C are similar to those deduced from the Gouy–Chapman/partition equilibrium approach. Overall, the agreement between the theoretical and experimental results, obtained for the three proteins, strongly supports that quantitative studies of the binding of water-soluble, globular proteins to neutral phospholipid membranes can be achieved using the thermodynamic approach, specially at low ionic strength. At high ionic strength, the thermodynamic approach does not completely predict the shape of the curves, at high surface coverage, which might be attributed to the fact that it has been assumed an average value of zþ p in the calculations as representative of the whole isotherm. At low ionic strengths the zþ p values in each point of the isotherm are similar and, consequently, an average < zþ p > value can be adequate. However, at high ionic strengths, the zþ p values calculated

for every experimental data in the corresponding isotherm slightly vary, and the average zþ p value could be considered as a less representative parameter of the whole isotherm, which could be cor-

Table 3 Mean average values of the actual protein charge in solution zþ p , the molar free  0;L and in the lipid phase DG  0;A per energies of the protein in the aqueous phase DG P P mole of protein ions, the theoretical partition coefficient C and the effective interfacial charge m for the binding of protein to phospholipid vesiclesa. System

Lysozyme/PC < zþ p >  0;A (J mol1) 104 DG P 4  0;L (J mol1) 10 DG P 5 10 (M1) 5 10 (M1) Lysozyme/PC/PG (90:10) < zþ p >  0;A (J mol1) 104 DG P  0;L (J mol1) 104 DG P 5 10 (M1) 5 10 (M1) Myoglobin/PC < zþ p >  0;A (J mol1) 104 DG P  0;L (J mol1) 104 DG P 5 10 (M1) 5 10 (M1) Myoglobin/PC/PG (90:10) < zþ p >  0;A (J mol1) 104 DG P  0;L (J mol1) 104 DG P 5 10 (M1) 5 10 (M1)

pH 4.0

7.0

9.0

5.35 69.62 73.05 0.23 0.23 1.61

5.21 65.58 69.21 0.53 0.53 1.60

5.20 65.36 69.00 0.55 0.55 1.60

5.25 66.58 70.16 0.43 0.43 1.60

4.98 59.19 63.15 2.03 2.03 1.60

5.02 60.33 64.23 1.59 1.59 1.59

5.24 60.04 64.20 4.62 4.62 1.73

5.55 68.21 71.96 0.88 0.88 1.70

5.73 73.29 76.79 0.32 0.32 1.69

5.56 68.72 72.45 0.79 0.79 1.69

5.56 68.66 72.39 0.80 0.80 1.71

5.81 75.58 78.97 0.20 0.20 1.69

a In all calculations eL was 20.0 and it was considered that the protein is solvated in both aqueous and lipid phases.

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 0;A and rected by introducing, in the theoretical expression of DG P 0;L  DGP , additional terms accounting for the interaction between the free protein and the counterions present in the solution. Table 3 indicates the average < zþ p > values, obtained for the proteins upon binding to phospholipid SUVs at various pHs. The < zþ p > slightly decreases as pH increases, because of the acid–base reaction of the amino acids that constitute the proteins. We obtain þ zþ p lower than the electrostatic charges. Once zp is known the free  0;A and lipid molar energies of the proteins, between the water DG P  0;L , have been estimated vs. pH and C values. Both phases DG P  0;A and DG  0;L display negative values, and their absolute values DG P P decrease as pH increases. At first glance, the fact reveals that the thermodynamic process of dissolving the proteins, in both phases, is spontaneous as observed for the three proteins. The differences  0;A  DG  0;L are positive values and slightly increase with pH, DG P P denoting a more favourable thermodynamic process of the proteins to be dissolved in the lipid phase. Notice that for a given pH, the values of C are similar to those deduced from the Gouy– Chapman/partition equilibrium approach. Some of our calculations, assuming the formal negative charge zL for slightly anionic SUVs and high surface coverage, did not converge because the Gouy–Chapman model is limited to low coverage, but a high surface concentration of cationic ad-molecules would cause zL to become less negative. In these cases the problem has been overcome allowing zL to be a fitting parameter. 4. Conclusions From the present results and discussion the following conclusions can de drawn. 1. It has been shown the major role of electrostatics, in the adsorption process of three relatively hydrophilic globular proteins and in the physicochemical stability of vesicles adsorbed with proteins. Adsorption to anionic vesicles occurred only when the mean charge of the proteins was positive. Upon protein adsorption the charge on the vesicles was reduced. Consequently the driving force for further surface coverage decreased. For lysozyme and myoglobin the extent of charge neutralization, determined by spectrofluorimetry, could be quantitatively ascribed to the mean charge of the proteins. The blue shift, in the fluorescence spectra of proteins, is associated with conformational changes and consequent exposure of aromatic amino-acid residues. 2. Lysozyme association with PC/CL membranes resulted in the gradual decrease of fluorescence, with protein concentration. The ionic-strength dependence of the effect suggested the substantial contribution of electrostatic interactions to detected bilayer modifications. The initial binding step of lysozyme to the surface of the lipid membrane is governed by electrostatics. Subsequent to the initial stage of binding, changes of the structure of the protein and lipid components of the membrane occur, leading to a new and more complex situation, involving the penetration of lysozyme into the lipid phase. 3. For lysozyme and myoglobin, an excellent correlation is found between effective charge and mean protein charge. The knowledge of the molecular details of the interaction between lysozyme and membrane phospholipids is of interest for two reasons. (1) Lysozyme provides a convenient and suitable model for studying lipid–protein interactions, involving initial electrostatic binding followed by putative penetration into the lipid bilayer. (2) The lysozyme–lipid interaction may provide important clues to understand bactericidal action. Lysozyme, which is positively charged at pH 7.0, produces the aggregation and fusion of negatively charged SUVs. These results are consis-

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tent with the ability of electrostatic charges, in protein binding to lipid SUVs, to produce the phenomena. The protein acts via a conformational change produced upon binding to the lipid SUVs, involving an increase in loops and unordered structure, at the expense of b-sheet conformation and b-turns. The forces involved in the interaction of lysozyme with lipid SUVs are not only electrostatic. In addition, membrane destabilization also plays a role in the aggregation and fusion of lipid SUVs, induced by proteins. Lysozyme binding was demonstrated to alter the structure of the lipid phase of negatively charged membranes. Some results indicate the existence of hydrophobic interactions between the protein and the lipid bilayer. Strong experimental support, for a hydrophobic component in the interaction, is provided by the finding that not all bound lysozyme can completely dissociate from such membranes, by either increasing the ionic strength of the buffer solution or dilution of the bulk protein. Consistent with the electrostatic nature of the interaction, the effect occurs despite the fact that the initial binding itself is rather sensitive to both ionic strength of the solution and amount of charge in the lipid membrane. The fact that electrically neutral lipid SUVs are able to bind lysozyme evidently indicates an affinity, based on hydrophobic interactions. Electrostatic interactions between the positive charges of lysozyme and negatively charged SUVs do also occur. 4. Lysozyme (pH 7) showed a significant electrostatic selectivity for anionic phospholipid species (PC < PG < PS < PI < CL), especially at low ionic strength, as can be expected for a cationic protein and anionic phospholipids. The corresponding interpretation is that the electrostatic interactions dominate over the hydrophobic ones. The electrostatic selectivity (PC < PG < PS) is in agreement with Förster resonance energy transfer (FRET) studies between cationic surfactant protein B (SP-B) of pulmonary surfactant and anionic phospholipids [79–81]. On the other hand, the selectivity of myoglobin (pH 4, low saline concentration) is the contrary (PS < PG < PC), confirming that lipid–myoglobin interactions are dominated by the hydrophobic interactions, in agreement with experiments with cationic surfactant protein C (SP-C) and anionic phospholipids [79–81]. 5. The agreement between the theoretical and experimental results, obtained for the three proteins, strongly supports that quantitative studies of the binding of water-soluble, globular proteins to neutral phospholipid membranes can be achieved using the thermodynamic approach. However, at high ionic strength, the thermodynamic approach does not completely predict the shape of the curves at high surface coverage, which might be because it has been assumed an average value of zþ p in the calculations as representative of the whole isotherm. The zþ p values calculated for every experimental data in the corresponding isotherm slightly vary, and the average zþ p value could be considered as a less representative parameter of the whole isotherm, which could be corrected by introducing, in the theoretical expression of the free energies of the protein in the aqueous and lipid phases, additional terms accounting for the interaction between the free protein and the counterions present in the solution. Work is in progress to give a better quantitative description of the association isotherms, at high ionic strength, and to improve the approach for negatively charged lipid membranes. 6. With the Gouy–Chapman formalism c is obtained as ln c / m sinh1(m)  m2. The activity coefficient goes with the square of the charge number. Thus for an effective charge reduction by a factor of 3 (meff = m/3), e.g., melittin [36], the corresponding reduction in the interaction energy is by a factor of 9. As C / c at constant m it can be expected ln C / ln c / zLsinh1(zL)  zL2. Further work will deal with the

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asymmetric location of the anionic phospholipid, in the inner leaflet of the bilayer in mixed zwitterionic/anionic SUVs, for the lysozyme–PC/PG and myoglobin–PC/PG systems.

Acknowledgment The authors acknowledge financial support from the Spanish DGES (Project No. MAT2006-03997). References [1] [2] [3] [4] [5] [6]

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