Binding of globular proteins to lipid membranes studied by isothermal titration calorimetry and fluorescence

Binding of globular proteins to lipid membranes studied by isothermal titration calorimetry and fluorescence

Colloids and Surfaces B: Biointerfaces 24 (2002) 53 – 61 www.elsevier.com/locate/colsurfb Binding of globular proteins to lipid membranes studied by ...

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Colloids and Surfaces B: Biointerfaces 24 (2002) 53 – 61 www.elsevier.com/locate/colsurfb

Binding of globular proteins to lipid membranes studied by isothermal titration calorimetry and fluorescence Mariana N. Dimitrova a, Hideo Matsumura a, Nelly Terezova b, Vassil Neytchev b,* b

a Electrotechnical Laboratory, AIST, MITI, Tsukuba 305 8568, Japan Institute of Biophysics, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria

Received 2 April 2001; received in revised form 30 May 2001; accepted 30 July 2001

Abstract The interactions of bovine serum albumin, lysozyme and cytochrome c with phosphatidylcholine liposomes in liquid crystalline phase have been investigated using isothermal titration calorimetry in combination with steady state, and fluorescence measurements. Calorimetric titration studies of the binding of liposomes to the protein species indicate in all cases exothermic processes with single binding sites in the protein molecule. Distinct saturation of the protein–lipid binding processes was observed at high molar lipid/protein ratio of 200/1. The thermodynamic parameters, including association enthalpy and entropy, Gibbs free energy and binding constant were determined at a constant temperature of 30 °C. Another set of experiments was performed using a fluorescence probe to determine the membrane order parameters such as limiting anisotropy, differential tangent and emission lifetime of fluorophore. The binding of proteins to liposomes significantly alters all membrane order parameters. The thermodynamic and fluorescence data presented indicate differences in the binding behaviour of these proteins to liposome suspensions. Information about thermodynamic properties of binding of globular proteins to liposomes and protein-induced membrane ordering can thus provide some clues about the nature of the protein– lipid complexes. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Globular proteins; Liposomes; Binding; Isothermal titration calorimetry; Fluorescence

1. Introduction Binding between proteins and lipid assemblies as liposome suspensions is a particularly interesting case and sometimes has a complicated energy * Corresponding author. E-mail address: [email protected] (V. Neytchev).

profile. This process involves different energetic expenditures for the adsorption of protein molecule at lipid interface, or for its partial or complete penetration into bilayer [1–7]. The free energy of complex formation is small due to the delicate balance between large favourable and unfavourable contributions. Moreover, binding takes place only if the total free energy change

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decreases, regardless of the actual amount of favourable free energy change accumulated by direct molecular contact between the associating biomolecules. This is to say, solvent effects are as important to the energy balance as are direct noncovalent interactions [8]. Previous reports have shown that above certain effective concentration (10 − 1 mg ml − 1) soluble globular proteins such as bovine serum albumin (BSA), lysozyme (LSZ), and cytochrome c (CC) demonstrate similar behaviour in the case of proteininduced flocculation of phospholipid liposomes [9,10]. The adsorption of BSA and LSZ on the liposomes caused membrane destabilization followed by leakage of the inner liposome content; meanwhile CC which adsorb mainly due to the electrostatic attraction may induce bridging flocculation. These globular proteins bind comparatively easily to large unilamellar phospholipid liposomes made from synthetic saturated lecithins. BSA (negative charge at pH 7.5) and LSZ (positive charge at pH 7.5) have loosely folded structures in aqueous solution and may exist in a well compact form as globes surrounded by a hydration shell. CC is a cationic peripheral membrane protein and is not loosely folded in water solutions. It is very stable at pH 7.5. However, it is of interest for membranology to know the specific differences in the intimate interaction mechanisms of individual proteins with lipid suspensions. This is likely to lead to differences in the nature of individual proteins, which should reflect on the binding parameters of protein–lipid interaction. The objective of this work is to elucidate the globular protein–phospholipid binding by investigating the association of BSA, LSZ, and CC with model phospholipid membranes, at temperature above the phase transition temperature of the phospholipid and neutral pH. Special attention is paid to the protein-induced membrane structural arrangement due to the differences in protein binding to the lipid bilayer. A thermodynamic description and analysis of the individual thermodynamic parameters of these processes is also given.

2. Materials and methods

2.1. Materials The proteins and lipids used were commercial products purchased from Sigma Chemical Co. Membrane forming lipid phosphatidylcholine (PC) was used without further purification. The fluorescence marker 1,4-(trimethylammonio)phenyl-1,6-diphenyl-1,3,5-hexatriene (TMA– DPH) was purchased from Boeringer Mannheim (Germany). Solutions were prepared using freshly distilled and deionised water obtained by Autostill system (WG 240 Yamato Co., Japan).

2.2. Liposome preparation Dispersions of well defined large unilamellar liposomes were prepared by means of the extrusion method, reported elsewhere [11]. Briefly, 100 mg of the PC, dissolved in n-hexane was dried under nitrogen flow. A volume of 4 ml of buffer solution containing 20 mM NaCl and 10 mM Tris–HCl (pH 7.5) was added to the experimental tube. The liposome dispersions were formed by a thorough vortexing for 20–60 min at room temperature and then followed by extrusion through 100 nm polycarbonate filter (Nucleopore Corp., Pleasanton, CA) under 200 psi of nitrogen. Bartlett assay was used to determine the molar concentration of PC in the final volume [12]. The size distribution of liposome particles in the dispersions was monitored experimentally by means of a dynamic light-scattering size-meter (Photal Otsuka Electronics DLS 800, Japan, Co. Ltd). The mean radius of PC liposomes in ITC and fluorescence experiments was 1209 27 nm (n= 6) and confidence limits of 95%.

2.3. Isothermal titration microcalorimetry The enthalpy change of protein–lipid associations was measured in an Omega reaction cell with a Microcal high-sensitivity titration colorimeter (Microcal, Northampton, MA) at 30 °C. The calorimeter was calibrated electrically as described previously [13]. Solutions were thoroughly degassed for 2 h under vacuum prior to usage.

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The protein solutions were placed in the 1.3684ml cell and the liposome dispersion in a 250-ml syringe at total molar concentrations of 15× 10 − 3 mM and 4.2 mM, respectively. In the experiments performed, 32 aliquots of volume 8.017 ml liposome dispersion were injected in the experimental cell. Time between each injection in one measurement was 4 min and the aliquots were added at a rate of 1 ml/2 s. The heat associated with the interaction of injected liposome dispersion with the protein solution was recorded to obtain the enthalpy binding isotherm. The heat of dilution of the liposome suspension was subtracted from the measured heat of mixing. The protein/PC interactions were analysed with a onesite binding model by ORIGIN software programme provided by Microcal Inc. The reproducibility of the binding enthalpy was in the order 9 0.2 kcal mole protein − 1.

2.4. Fluorescence measurements Steady state fluorescence intensity and anisotropy (SSFA) experiments were performed as described previously [14]. Our purpose here is to examine the effects of different proteins on the structural order of lipid bilayer and by this way to get additional information concerning protein– lipid binding. Fluorescence marker with concentration of 60 mm in water was added to the stock suspension of reconstituted liposomes containing different proteins in a molar ratio lipid/protein 50:1. The lipid concentration in the liposome suspension was as in ITC experiments equal to 4.2 mM. The reconstituted liposomes were obtained using the same procedure for preparation of liposomes as described above. The experiments were performed at constant temperature 30 °C, in a Perkin –Elmer MPF3 spectrophotometer with polarisers in excitation and emission beams. The fluorescence intensities parallel and perpendicular to the excitation beam were recorded automatically. The excitation wavelength was 360 nm while the detection was at 430 nm. The limiting anisotropy, r , which is related to the molecular orientation of the probe was calculated using the following equation:

r= r + (r0 − r)/6R~

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(1)

where r is the experimentally measured fluorescence anisotropy, r0 is the maximum anisotropy in the absence of rotational diffusion, ~ is the emission lifetime, and R is the rotational rate of fluorophore. It is usually assumed that the lifetime is a constant value in homogenous lipid assemblies. A plot of (r0 − r)/~ yields a slope of (6R) − 1 and an intercept of r . The value of ~ was obtained separately from emission lifetime fluorescence (ELT) measurements as reported in our previous work [14]. In these measurements the samples were excited with sinusoidally modulated polarized light, and the phase difference between the perpendicular and parallel component of the emission was simultaneously measured with two detectors. The emission lifetimes of 60 mm fluorescence probe in ethylene glycole were measured to obtain the mean value of ~. This method has been reported in detail in Ref. [15], and enables us to determine important structure order parameters by the combined use of SSFA and ELT measurements. To determine the fluorescence intensity in the presence of CC a correction for inner filter was performed as given in literature [16].

3. Results and discussion

3.1. Thermodynamic parameters of the protein– PC liposome interactions The binding properties of three globular proteins, BSA, LSZ, and CC with PC liposomes in buffer solution at pH 7.5 were consecutively studied using an isothermal titration calorimetry method. The total heats produced exothermally while injecting small aliquots of liposome dispersions in the protein solutions at constant temperature 30 °C were registered, as shown in Fig. 1(A–C). Measurements of this heat allows the very accurate determination of the thermodynamic profile of the molecular interaction in a single experiment. At this temperature the heat of dilution of the liposome dispersion from the syringe into calorimeter cell was less than 0.059 0.01 kcal mole − 1 and was subtracted from the

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raw ITC data. It was observed that after the first several injections of liposome dispersion into the ITC cell, the released heat decreased differently in

the individual samples. The exothermic peaks are of variable magnitude over all injections. A distinct difference in the saturation of the exothermic

Fig. 1. Isothermal heat capacity curves of the PC liposomes with BSA (A), LSZ (B), and CC (C) at 30 °C neutral pH. The molar concentration of the proteins in the cell (volume 1.4 ml) was 15 × 10 − 3 mM. The PC liposome molar concentration in the syringe (volume 0.25 ml) was 4.2 mM. Injections scheduled as described in the text.

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Fig. 1. (Continued)

reactions could be distinguished in the curves of DH as a function of the lipid/protein molar ratio for the three samples. In the cases of BSA and LSZ the saturation was reached at lipid/protein molar ratio 200/1. For CC the saturation was reached at lipid/protein molar ratio higher than 300/1. After the saturation, the interactions were independent of molar ratio. The variations of the released heats shown in Fig. 1(A– C) were in a wide range from 2 to − 8 kcal mole − 1 of the injectant, which may serve as an indication for specific differences in the interaction mechanism of individual reactions. Analysis of binding isotherms permitted the main binding parameters to be fitted as a function of the number of binding sites, j, in the protein molecule. These are summarized in Table 1. The values of DH (enthalpy of association) and binding constants K were estimated for all samples on the bases of the best fit of the integrated heat effects using a non-linear least squares method. The enthalpies of association were negative at one binding site, and significantly different from − 73.14 to −667.78 kJ mole − 1 typical for the injectant. They increased in

the order: LSZB BSA B CC. We analysed the negative sign of enthalpy changes as an eventual possibility that hydrophobic bonds may be formed during the interaction of PC liposomes with three globular proteins. In this study BSA and LSZ have similar values of DH. This fact is of particular interest, since these proteins demonstrate a significant penetration into lipid bilayer Table 1 Thermodynamic parameters for the interaction of BSA, LSZ and CC with PC liposomes obtained in ITC experiments, at 30 °C Parameters

j K (M−1) DH° (kJ mol−1) DG° (kJ mol−1) TDS° (kJ mol−1)

Protein BSA

LSZ

CC

1.14 5010 −79.12 −21.46 −57.67

1.08 760 −73.14 −16.69 −56.44

1.02 70.7 −667.78 −10.71 −657.07

The experimental concentrations are the same as described in Fig. 1. j denotes the number of binding sites in protein molecule.

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due to the increased hydrophobic intermolecular interactions [10]. CC’s side chain penetration into the interface region of the bilayer probably occurs in addition to a bilayer surface electrostatic interaction between the lipid’s negative head groups and the protein dipole moment arising primarily from the accumulation of lysine residues around the protein’s hem cleft [17,18]. Nevertheless, we suppose that the most significant part of the heat is due to the protein adsorption into bilayer [9,10]. It is seen from Table 1 that the values of K increase in the order CCB LSZ BBSA. In the case of LSZ and BSA they are 10– 70 times higher than the corresponding K for CC. Our data confirm the conclusion of some authors that such complex interactions which involve hydrophobic and weak electrostatic forces lead to low values of K. Moreover, the low affinity binding may be associated with conformational changes in the protein molecule [19]. The structural transformations and differences in the side chain groups of the individual proteins also may affect the binding parameters. This finding is in fair agreement with a previous report showing that the higher binding affinity of BSA with PC liposomes in liquid crystalline phase may be a consequence of the greater tendency of these molecules to associate especially with well defined and structured PC bilayers [20,21]. In these publications BSA is classified as ‘‘softprotein’’, easy to perturb the lipid surface. The higher values of K for BSA and LSZ may also be due to their greater tendency to self associate [22,23]. Regarding LSZ it is known that the nature of cationic interactions with zwitterionic lipids such as PC strongly influences the macroscopic properties of the lipid– protein complex. The cationic reaction in this case can alter the nature of hydrogen bonding at the membrane surface. The latter relates to additional hydrophobic dehydration interactions between the hydrophobic side chains of LSZ molecule and PC membranes. We also found a clear expression of the Gibbs free energy change, DG°, in the association reactions. In general, changes of DH° are not correlated to changes in DG° [8]. One reason for this

unfortunate situation is enthalpy/entropy compensation. The overall change in binding enthalpy due to a particular mutation can be partly compensated by an entropy change. As a result, there is only a small change in the free energy of binding DG°, as can be seen from the data in Table 1. The data increased in the order: CCB LSZ BBSA and correlate well with free energy of unfolding of these proteins. CC is known as one of the most stable proteins in water solutions at neutral pH. In contrast, the changes in the entropy of binding DS° are largely caused by hydration effects because the entropy of hydration of polars and apolars groups is large and there is a significant reduction of water accessible surface on binding. In all cases the values of DS° are negative and they indicate that hydrophobic interactions do not dominate the binding process. The reduction of entropy has different reasons, apart from the hydration of the surface, which remains unchanged or increased with respect to the free partners [8]. Another important contribution to DS° may originate from the changes of side chain mobility at the binding site of lipid–protein complex. This should affect the respective molecular configuration of the adsorbed protein molecules. It is not surprising since the minimum value of TDS°= 657.07 (kJ mol − 1) was found for CC-lipid binding, showing that the association in this case is more unfavourable in comparison to LSZ-lipid and BSA-lipid associations. These data are in fair correlation with a number of previous reports considering the adsorption mechanisms of these proteins [24,25]. It is considered that the association of CC to lipid membranes is due to long-ranged electrostatic interactions, while BSA and LSZ association is caused by complex (electrostatic, and short ranged hydrophobic) interactions. In the case of BSA it is known that this protein may strongly absorb deeply into bilayer due to hydrophobic dehydration forces acting at a very close protein–membrane approach. However, the entropy data for LSZ and BSA shown in Table 1 are very close, and it can be assumed that the changes in heat capacities accompanying both processes of association are similar.

M.N. Dimitro6a et al. / Colloids and Surfaces B: Biointerfaces 24 (2002) 53–61 Table 2 The membrane structural order parameters in the binding of proteins to PC liposomes at 30 °C Structural order parameters

Control Sample 1. LSZ 2. BSA 3. CC

r

~0 (ns)

tan Z

0.0547 90.0002

6.411 9 0.005

0.0574 9 0.0005

0.0764 90.0005 0.0875 90.0008 0.1318 90.0026

4.805 9 0.007

0.0421 9 0.0013

4.487 9 0.009

0.0390 9 0.0015

3.432 9 0.007

0.0306 9 0.0007

Errors are 9SD in triplicate determinations.

3.2. Binding of proteins to liposomes and membrane-ordering To determine the role of globular protein-induced structural changes on the lipid membrane, we also found the values of structural order parameters as described previously [14]. They were obtained from fluorescence polarization and anisotropy measurements in the binding process. The most popular fluorescence probe used in such case is TMA– DPH which is a polyene hydrocarbon with an elongated rectangular shape and a stable all-trans configuration. The motional properties of this probe depend mainly on the lipid environment and the arrangement of the polar head groups at the lipid surface [15]. In the same time, the fluorescence properties of the TMA– DPH vary when the probe molecules are inserted at specific positions in fatty acids. Using this probe the structural order parameters in PC liposomes can be determined. Table 2 gives the mean values for the structural order parameters of the studied samples compared to the control (liposomes without protein). As a rule it is known that in homogeneous liquid– crystalline lipid phase, the adsorption of proteins to the lipid membrane promoted apparent ordering which is indicated by the increase of r , respectively, decrease of ~ and tan Z in comparison to the control. As can be seen from the data in Table 2, all samples studied

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showed an increase of r against the control which may be considered as an apparent structuring of lipid membrane under association with three proteins. A maximum value for r was observed in the sample for CC, which is nearly 100% higher in comparison to the control. The determination of parameter r can be used to define the degree to which the fluorophore rotation was hindered. In the same time the values of r for LSZ and BSA changed between 35 and 40%, indicating lower levels of structuring of lipid membrane than in the case with CC. On the contrary, the values for ~ and tan Z were found to decrease to within 30 and 50% against the control. The globular protein effect on the membrane ordering was investigated by combination of SSFA and ELT fluorescence methods. These methods are successfully applied in the study of lipid–protein interactions. Membrane ordering and surface domain formations are well established processes, and have been shown to be of importance to the lipid–protein binding. The mismatch between the lengths of the hydrophobic membrane spanning segments of penetrating protein and those of lipids was proposed as a mechanism causing structural changes of lipid membrane [26]. Hydrophobic matching conditions in lipid–protein binding have also been shown to induce membrane ordering in fluid liquid crystalline phospholipid liposomes [27]. From this point of view it should be of interest to compare the thermodynamic parameters of lipid– protein association with those responsible for membrane ordering obtained in fluorescence experiments. The addition of protein to the liposomes significantly alters all measured quantities in fluorescence experiments. The increase of r in different samples was related to the way the proteins may affect the membrane structural order [28]. Our results are compatible with this interpretation, moreover that these data are confirmed by the measured ~ and tan Z. There was significant effect of CC on ~. The TMA –DPH lifetime decreased from 6.411 in the control to 3.432 ns in sample suggesting strong modification of the binding location and rotational characteristics of the membrane bound TMA–DPH in this case. The results of this work showed once more

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that TMA –DPH binding to lipid membranes is obviously associated with membrane ordering. Some caution is required when interpreting the effects these parameters have on membrane ordering S. Confusion exists due to the different techniques used to measure different parameters, the inappropriateness of some of these parameters as indicators of membrane ordering and, in some cases, the disagreement of their measured values. Furthermore, the membrane ordering parameter S is related to the reciprocal of the viscosity in bulk phase and varies when probe molecule is bound at specific positions in fatty acids, or stays at membrane surface. The nature of S is thus defined and may give us important information about the structural characteristics of lipid membranes in the presence of globular proteins.

4. Conclusions The ITC experimental data, presented in this study, indicate differences in the basic thermodynamic parameters describing binding phenomena of three globular proteins BSA, LSZ and CC with PC liposome suspensions in liquid crystalline phase. There are distinct differences in the total enthalpy and entropy changes of BSA and LSZ from one side and CC. It was found that the association of these proteins to PC membranes is a one-step exothermic process, which, despite the unfavourable entropy change, brings about the release of an essential amount of reaction heat. The lipid/protein binding involves one binding site in the protein molecules at a comparatively high molar ratio, and this finding correlates well with recent reports concerning the number of binding sites in similar interactions [24,25]. We also believe that the exotherms observed for these reactions are caused by weak associations of protein and lipid since the processes reach saturation at high values of the molar ratio. In the same time these weak associations may influence the membrane lipid structuring depending on the type of protein and its ability to adsorb on the lipid

membrane. Entropy losses in the binding process are anticipated since the mobility of proteins as well as the undulations of the lipid membrane are significantly reduced due to the protein adsorption. Our fluorescence data confirmed this assumption. The entropy changes are in fair correlation with the degree of membrane structuring. For example, the association of CC has been considered by many authors [21,29,30] as a paradigm of an electrostatic binding of peripheral protein to lipid bilayer due to the longranged electrostatic forces with following limitation in membrane mobility. The last was interpreted as an increase of membrane structuring. On the contrary, the association of LSZ and BSA is favoured by significantly higher entropy changes due to the short-ranged hydrophobic interaction with eventual penetration of these proteins into lipid membrane. The protein molecules may cause apparent disordering of lipid membrane, and the association process is thermodynamically more favourable. Although the thermodynamic parameters found in this study do not directly lead to a molecular model, they correlate well with the data for membrane ordering in the process of lipid/protein interactions, which allowed us to relate the influence of these proteins to the membrane ordering. Finally, by direct measurements of the enthalpy changes on binding and determination of membrane order parameters, it is possible to indicate where the protein binds to a phospholipid liposome. Information about thermodynamic properties and ordering of lipid membranes in the process of binding of proteins can thus provide some clues about the nature of lipid–protein complexes.

Acknowledgements This work was partially supported by a research fellowship awarded to M.N.D. by the agency of Industrial Science and Technology, Japan. The authors are grateful to Professor B. Djakov from Bulgarian Academy of Sciences for the challenging discussions and critical reading of the manuscript.

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