Effect of Amino Acid Sequence Change on Peptide−Membrane

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Effect of Amino Acid Sequence Change on Peptide-Membrane Interaction P. Sospedra,† C. Mestres,† I. Haro,‡ M. Mun˜oz,† and M. A. Busquets*,† Department of Physical Chemistry, Faculty of Pharmacy, University of Barcelona, Avgda Joan XXIII, s/n, 08028 Barcelona, Spain and Department of Peptide & Protein Chemistry, IIQAB.CSIC, Jordi Girona Salgado, 18, 08034 Barcelona, Spain Received July 24, 2001. In Final Form: November 3, 2001 The RGD region of the (110-121) peptide sequence (FWRGDLVFDFQV) of VP3 capsid protein of hepatitis A virus, which is described to be responsible for a high immunoresponse, was changed for RGE and RKD amino acids in order to analyze the change effect on the physicochemical properties of the peptide. Peptides were synthesized by solid-phase synthesis and characterized by amino acid analysis, analytical highperformance liquid chromatography, and electrospray mass spectrometry. The peptides had surface activity concentration dependent, formed stable monolayers at the air/water interface, and were able to insert into lipid monolayers of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), 1,2-dipalmitoyl-sn-glycero3-phosphatidylglycerol, phosphatydic acid, L-R-phosphatidyl-L-serine, stearylamine, sphyngomyelin, and liver extract. Differential scanning calorimetry (DSC) was used to investigate the thermotropic phase properties of binary mixtures of DPPC and the three peptides. DSC data showed a complete abolition of DPPC pretransition and significant broadening of the main phase transition with increasing amounts of peptide. These results are indicative of an interfacial location of the peptides and with some penetration of the nonpolar amino acid side chains into the hydrocarbon chain region closer to the polar/nonpolar interface. Finally, fluorescence spectroscopy confirmed the preferential interaction of the peptides with the liquid crystalline state of the bilayer with a contribution of both hydrophobic and electrostatic forces.

1. Introduction The penetration of the nonenveloped Hepatitis A virus (HAV) in a cell remains an unclear process. It is related to the presence of the proteins known as VP1, VP2, VP3, and VP4 located at the capsid of the virus. Such epitopes can be mimicked by means of synthetic peptides, and some of them have proved to be highly immunogenic.1-3 An approach to get information about the potential immunoresponse of the peptides is the analysis of their physicochemical properties. In that sense, several investigations have been done studying the effect of chain length, charge, and amino acid substitution on the physicochemical properties of the native protein.4-5 For instance, from these studies it has been concluded that shorter analogues of a continuous epitope of hepatitis A virus, VP3 (110-121) peptide, failed to react with convalescent sera, indicating the importance of the entire peptide in the epitope structure.2 The comprehension of the interaction of the antigenic peptides with phospholipid membranes is important not only to gain insight into the infection and proliferation mechanism of Hepatitis A virus but also to understand, in view of its possible targeting, the effectiveness of these * To whom the correspondence should be addressed. Phone: +34-93-4024556. Fax: +34-93-4035987. E-mail: busquets@ farmacia.far.ub.es. † University of Barcelona. ‡ Department of Peptide & Protein Chemistry, IIQAB.CSIC. (1) Haro, I.; Pinto´, R. M.; Gonza´lez-Dankaart, J. F.; Pe´rez, J. A.; Reig, F.; Bosch, A. Microbiol. Immunol. 1995, 39, 845. (2) Bosch, A.; Gonzalez-Dankaart, J. F.; Haro, I.; Gajardo, R.; Pe´rez, J. A.; Pinto´, R. M. J. Med. Virol. 1998, 54, 95. (3) Pinto´, R. M.; Gonzalez-Dankaart, J. F.; Sa´nchez, G.; Guix, S.; Go´mara, M. J.; Garcı´a, M.; Haro, I.; Bosch, A. FEBS Lett. 1998, 438, 106. (4) Pe´rez, J. A.; Canto´, J.; Reig, F.; Pe´rez, J. J.; Haro, I. Biopolymers 1998, 45, 479. (5) Sospedra, P.; Mun˜oz, M.; Garcı´a, M.; Alsina, M. A.; Mestres, C.; Haro, I. Biopolymers 2000, 54, 477.

peptide-loaded liposome formulations. With a focus on VP3-related peptides, the most favored interaction with biomembrane models was obtained with the VP3 (110121) peptide and its analogue VP3 (110-119).4 In particular, the Arg-Gly-Asp (RGD) triad present in position 112-114 of the (110-121) peptide sequence, FWRGDLVFDFQV, of VP3 capsid protein of hepatitis A virus, is described to play an important role in generating immuno response.2 In addition, it is necessary to induce the β-structure in the presence of liposomes. This RGD sequence has also been described to be of great importance as a universal recognition site.7-8 For instance, it plays a relevant role in the inhibition of fibrinogen binding to platelet RIIbβ3 by inducing an allosteric change in the amino-terminal portion of RIIb.9 The aim of the present investigation is to experimentally test the effect of the replacement of RGD sequence by RGE and RKD in the membrane binding potential and to discuss their role as likely antibody generating agents. In that sense and taking into consideration that unraveling the peptide-lipid membrane interaction on a molecular level is a prerequisite for understanding virus proliferation and infection process, we have considered the use of model membranes to get insight in such a process. Lipid monolayers and liposomes are suitable models to get information about physicochemical data needed to predict the forces involved in the interaction. They are useful tools to investigate molecule-membrane interactions at different depths and to gain information on the effect produced by the molecule on lipid bilayer packing and (6) Pe´rez, J. A.; Gonza´lez-Dankaart, J. F.; Reig, F.; Pinto´, R.; Bosch, A.; Haro, I. Biomed. Pept., Proteins Nucleic Acids 1995, 1, 93. (7) Pierschbacher, M. D.; Ruoslahti, E. Proc. Natl. Acad. Sci. U.S.A. 1984, 81, 5985. (8) Pierschbacher, M. D.; Ruoslahti, E. Nature 1984, 309, 30. (9) Basani, R. B.; D’Andrea, G.; Mitra, N.; Vilaire, G.; Richberg, M.; Kowalska, M. A.; Bennett, J. S.; Poncz, M. J. Biol. Chem. 2001, 17, 13975.

10.1021/la011156v CCC: $22.00 © 2002 American Chemical Society Published on Web 01/26/2002

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Table 1. Amino Acid Sequence of the Peptides and Their Charges residues

name

VP3(110-121)

VP3

[Glu]114VP3(110-121)

A2

[Lys]113VP3(110-121)

A3

sequence

net charge

+ NEGATIVE FWRGDLVFDFQV + NEGATIVE FWRGELVFDFQV + +NEUTRAL FWRKDLVFDFQV

orientation. The Langmuir-Blodgett technique was used first to investigate the surface activity of the peptides or accumulation at the air/water interface and, second, to get insight about the ability of the peptide sequences to insert into lipid monolayers composed of 1,2-dipalmitoylsn-glycero-3-phosphatidylcholine (DPPC), 1,2-dipalmitoylsn-glycero-3-phosphatidylglycerol (DPPG), L-R-phosphatidyl-L-serine (PS), L-R-phosphatidic acid (PA), stearylamine (SA), sphingomyelin (SM), and liver extract. The last one was chosen because its composition is the most comparable to the hepatocyte membrane, the target cell of the virus. Interactions of molecules, in our case peptides, with a bilayer-ordered structure can influence vesicle transition thermotropic parameters according to their own physicochemical properties. To study this effect with the synthesized peptides, we used the differential scanning calorimetry technique (DSC) to analyze the effect of increasing amounts of peptide on the thermotropic properties of multilamellar vesicles composed of zwitterionic DPPC. Furthermore, we have employed fluorescence spectroscopy to monitor the peptide effect on small unilamellar vesicles (SUVs) of DPPC and DPPC/DPPG (95/5). 2. Experimental Section Chemicals. DPPG, PS, PA, SA, and SM were from Sigma. DPPC and liver extract were from Avanti Polar Lipids. Their purity, checked by thin layer chromatography, was of 99%. Lipids were used without further purification. Chloroform and methanol (pro analysis) were from Merck. Water was double distilled. For fluorescence measurement, HEPES (5 mM) buffer was used, and for monolayer studies, the aqueous subphase was phosphate buffered saline (PBS). For both buffers, pH 7.4, 15.4 mS/cm, and 313 mOsm/kg. Peptides Synthesis. Peptide sequence was chosen according to the semiempirical method of Chou and Fasman10 that predicts theoretically the secondary structure of the peptides. This method was applied by using the Peptide companion version 1.24 (Coshisoft/PeptideSearch) computer program. Then, the peptides (Table 1) were synthesized by solid-phase methodology following a Fmoc/tBut strategy as already described.4,6 Briefly, synthesis of VP3 was carried out manually on a p-benzyloxybenzoyl alcohol resin of 0.8 mequiv/g of functionalization by means of a diisopropyl carbodiimide/hydroxybenzotiazole activation. The synthesis of VP3 analogues was performed automatically on an Abimed AMS 422 synthesizer on a 50 mmol scale using an initial Fmoc-ValWang resin (alkoxybenzyl resin) of a functionalization of 0.6 mequiv/g. Throughout the syntheses 2-(1H-benzotriazole-1-yl)tetramethyluronium hexafluorophosphate/N-methylmorpholine agents of condensation were used. Final deprotection and cleavage of the peptides from the resin was achieved by trifluoracetic acid (TFA) with appropriate scavengers (H2O and ethanedithiol) at room temperature with occasional agitation. Crude peptides were purified by medium-pressure liquid chromatography on a C18-silica column equilibrated with 20% acetonitrile containing 0.05% TFA in water and eluted with a linear gradient of 20-50% acetonitrile containing 0.05% TFA in water at a flow rate of 2 mL/min. Eluted samples were detected at 225 nm. The fractions were analyzed by reverse-phase highperformance liquid chromatography (HPLC); fractions of high purity were pooled and lyophilized. Surface activity measurements to determine the equilibrium spreading pressure of the peptides were recorded by using a Langmuir film balance KSV5000, equipped with a Wilhelmy

platinum plate.11 Increasing volumes of a concentrated solution of the peptides were injected beneath the surface of a cylindrical Teflon trough (70 mL of capacity) through a lateral whole. During the experiments, the subphase was continuously stirred (SBS Instruments, Spain). The increase of surface pressure (∆π) with time was recorded until a steady-state value of ∆π was obtained. Interaction of peptides with lipid monolayers was monitored with the same Teflon trough described above. Lipid monolayers at the required pressure (5, 10, or 20 mN/m) were formed by spreading the lipid or lipid mixture from chloroform/ methanol (2/1, v/v) solutions, on PBS subphases. The peptide was injected beneath the lipid monolayer, and pressure increases produced were recorded for 2 h to ensure that equilibrium was reached. The peptide concentration was slightly lower than the concentration at which the equilibrium spreading pressure is achieved. The subphase was stirred continuously to ensure an homogeneous distribution and a good interaction of the peptide with the monolayer. ∆π was recorded as described before. Compression Isotherms. Compression experiments were performed in a Teflon trough (17 000 mm2 surface area, 1 L volume). The output of the pressure pickup was calibrated by recording the well-known isotherm of stearic acid, which is characterized by a sharp phase transition at 25 mN/m for a subphase of pure water at 20 °C. Peptide films were spread from chloroform solutions, and at least 10 min was allowed for solvent evaporation. Films were compressed continuously with an area reduction rate of 60 mm2/min. All samples were run at least three times in the direction of increasing pressure with freshly prepared films, and the reproducibility was (0.01 nm2/molecule. The subphase temperature was 21 ( 0.5 °C. In addition, and to assess the stability of the monolayers, the film was submitted to compression and decompression cycles. Liposome Preparation. For DSC analysis, multilamellar vesicles (MLVs) of DPPC were prepared as follows. Briefly, the lipid was dissolved in a Pyrex glass tube with a mixture of chloroform/methanol (2/1, v/v) and the tube was shaken. Similarly, to prepare peptide-loaded vesicles, suitable amounts of the peptides were solubilized together with DPPC to reach 10, 20, 30, or 40 mol % of peptide in the preparation. Solvent was removed under a nitrogen stream. The residual solvent was eliminated by storing the samples overnight under high vacuum in a vacuum oven at room temperature. MLVs were obtained by hydrating the lipid film with HEPES buffer, pH 7.4 and vortexing at 50 °C. Final lipid concentration was 4 mM. For fluorescence purposes, SUVs of DPPC or DPPC/DPPG (95/5) were prepared from MLVs as described previously. In that case, lipid concentration was 30 mM. The mixture was then sonicated in a G112SPIT bath type sonicator (Laboratory supplies, Hicksville, NY) above the gel-fluid transition temperature until a clear dispersion was obtained. Vesicle size was determined by dynamic light scattering with a Malvern II-C Autosizer at room temperature. The mean diameter was 50 ( 5 nm, and the polydispersity index was 0.1 ( 0.02. Phospholipid content was analyzed by phosphorus quantification as previously described.12 Differential Scanning Calorimetry. DSC experiments of DPPC MLVs were performed using a DSC 821E Mettler Toledo (Greifensee, Switzerland) calorimeter. Hermetically sealed aluminum pans (nominal volume 40 µL) were used. Reference and sample pan masses were always matched to within 5.5% total mass and usually to within 2.0%. Pans were loaded by adding 30 µL of DPPC vesicle suspension, corresponding to ∼0.13 mg of phospholipid. All samples were submitted to three heating/ cooling cycles in the temperature range of 0-60 °C at a scanning rate of 5 °C/min. Data from the first scan were always discarded to avoid mixing artifacts. The endothermal peak coming from the second scan of the control sample was used as a reference template. The instrument was calibrated with indium. Fluorescence Experiments. Fluorescence measurements were carried out at room temperature or 50 °C in PBS (pH 7.4) on an AB-2 spectrofluorimeter (SLM-Aminco, Urbana, IL) (10) Chou, P. Y.; Fasman, G. D. Annu. Rev. Biochem. 1978, 47, 251. (11) Verger, R.; de Haas, G. H. Chem. Phys. Lipids 1973, 10, 127. (12) Barlett, G. R. J. Biol. Chem. 1959, 234, 466.

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Table 2. Peptide Characterization peptide

amino acid analysisa

VP3

F ) 2.9(3); W n.d. R ) 0.9(1); G ) 0.9(1) L ) 1.0(1); V ) 2.1(2) D ) 1.9(2) Q ) 0.9(1) F ) 3.0(3); W n.d. R ) 1.1(1); G ) 1.0(1) E + Q ) 2.1(2); L ) 0.9(1) V ) 1.7(2); D ) 1.2(1) F ) 3.1(3); W n.d. R ) 1.2(1); K ) 1.1(1) D ) 1.9(2); L ) 1.0(1) V ) 2.0(2); Q ) 1.1(1)

A2

A3

net charge

HPLC (k′)b

ES-MSc

-1

14.4

1528.5

-1

15.8

1542.5

0

15.1

1599.6

a Theoretic values in parentheses. b High-performance liquid chromatography (HPLC) conditions: (A) H2O (0.05% TFA), (B) CH3CN (0.05% TFA).; gradient, 95% A to 5% A in 35 min; λ, 215 nm; flow 1 mL/min; Spherisorb C-18; 10 µm column; k′, capacity factor. c ES-MS, electrospray mass spectrometry.

equipped with a thermostable cuvette holder, with constant stirring. The excitation and emission bandwith were set at 4 nm each, the wavelength used corresponding to tryptophan (Trp) being 285 and 340 nm, respectively. Peptide was added from a standard solution to a final concentration of 2 µM, and titrated with SUVs. The lipid to peptide molar ratio was 100:1 in all cases. Suspensions were continuously stirred, and in the case of the experiments at 50 °C, the system was allowed to equilibrate for 5 min before recording fluorescence data. Fluorescence intensities were corrected for contribution of light scattering by subtraction of the appropriate vesicle blank and for the innerfilter effect13 by applying the equation

F ) Fm × 10(Aex+Aem)/2 in which F is the corrected fluorescence intensity, Fm is the measured fluorescence intensity corrected for the light scattering, and Aex and Aem are the absorbances measured at the excitation and emission wavelength, respectively. The absorbance of the peptide samples was measured by using a LKB-Biochrom Ultrospec II spectrophotometer at 285 and 340 nm.

3. Results and Discussion Peptide Synthesis and Characterization. The synthetic peptides described in the Experimental Section were successfully characterized by HPLC, amino acid analysis, and electrospray mass spectrometry, and results are shown in Table 2. Due to the fact that for the majority of identified proteins the only structural information available is the amino acid sequence, empirical rules for predicting the position of continuous epitopes in proteins from several features of the primary structure (i.e., hydrophilicity, amphiphaticity, segmental mobility, etc.) have been developed.14 For prediction of the position of continuous epitopes, the highest peaks appearing in hydrophilicity plots should be considered. It has been shown that methods for predicting the secondary structure of proteins can be used as predictors of antigenicity. Algorithms that predict the position of loops or turns at the same time predict regions of highest hydrophilicity. In this sense, when the Chou and Fasman10 semiempirical method was applied to the peptides, the conformational parameters calculated for this peptide sequence showed a preference for β-type structures (βsheet and β-turn) over an R-helix or random coil. The (13) Lakowicz, J. R. In Principles of Fluorescence Spectroscopy; Plenum Press: New York, 1983. (14) Synthetic polypeptides as antigens; Van Regenmortel, M. H. V., Briand, J. P., Mullser, S., Plaue´, S., Eds.; Elsevier: Amsterdam, 1998; Chapter 1.

Figure 1. Surface pressure, π, as a function of concentration for VP3 (110-121) (O), A2 (b), and A3 (9)

theoretical prediction of the secondary structure of the peptides indicates a putative candidate for an exposed antigen. However, it is widely known that short peptides have no preferred conformation in solution, and furthermore, their most relevant conformation may depend on the environment.4 Peptide Surface Activity. The surface activity of the peptides was determined by injecting different volumes of a concentrate DMSO solution of the peptides into the PBS subphase and recording pressure increases. The absence of activity of the DMSO solvent was previously controlled. The incorporation of the peptide to the air/ water interface was fast up to a saturation concentration. Pressure increase, ∆π, at the saturation point was ∼16 mN/m for all the peptides. The surface excess (Γ) and area/ molecule calculated according to ref 15 were also very similar for VP3, A2, and A3, being ∼10 × 10-7 mol/m2 and ∼1.6 nm2/molecules, respectively. These data have been described in the literature to be useful for characterizing the effect of varying specific amino acids within the sequence of a peptide composed of a hydrophobic segment from different kinds of proteins such as HAV16 or HIV.17 As a summary, Figure 1 shows the equilibrium surface pressure versus concentration for VP3 (110-121), A2, and A3 peptides. For the three peptides, the plot follows a common trend that can be split in two sections. The first one corresponds to a linear increase of the surface pressure, π, with log C from the surface pressure onset up to a peptide concentration of 1.5 × 10-7 M (surface pressures of 14.08, 16.72, and 15.6mN/m for VP3, A2, and A3, respectively). This behavior is typical for a peptide random coil conformation.18 In the second part of the plot, the slope of the curve changes. Then the increase in surface pressure versus concentration was not as significant as in the first section, which indicates a pKa shift due to peptide association.19 This statement does not agree with that obtained by applying the Chou and Fasman10 method, a fact that assesses the importance of the environment on peptide conformation. These results are indicative of a combination of an amphiphilic and hydrophobic nature of the studied peptides. (15) Sospedra, P.; Haro, I.; Alsina, M. A.; Reig, F.; Mestres, C. Langmuir 1999, 15, 5303. (16) Mota, F. M.; Busquets, M. A.; Reig, F.; Alsina, M. A.; Haro, I. J. Colloid Interface Sci. 1997, 188, 81. (17) Vidal, P.; Chaloin, L.; Heitz, A.; Van Mau, N.; Me´ry, J.; Divita, G.; Heitz, F. J. Membr. Biol. 1998, 162, 256. (18) Seelig, A.; Alt, T.; Ku¨rz, I.; Mutter, M. Peptides 1992; Scheiner, M., Eberle, A. N., Eds.; ESCOM Science Publishers B.V., ESCOM: Leiden, 1993; pp 113-114. (19) Seelig, A. Biochim. Biophys. Acta 1990, 1030, 111.

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Figure 2. Surface pressure increases after VP3, A2, and A3 peptide insertion into various lipid monolayers as function of initial surface pressure: (a) 5mN/m; (b) 10 mN/m, and (c) 20 mN/m.

Peptide Insertion into Lipid Monolayers. Penetration Kinetics at Constant Area. The interaction of A2 and A3 with monolayers composed of DPPC, DPPG, SM, PA, PS, SA, and liver extract was studied through penetration kinetics at constant area, as described before and compared to the parent compound, VP3.20 Peptide concentration in the subphase was 0.315 µM. Parts a-c of Figure 2 show the end point pressure differences for the initial lipid surface pressure (π0) of 5, 10, and 20 mN/ m, respectively. Plots were recorded during 2 h in order to ensure equilibrium. In all cases, the surface pressure increase was highly dependent on the initial surface pressure, and insertion became increasingly difficult with increasing packing density of the lipid molecules. The (20) Sospedra, P.; Alsina, M. A.; Haro, I.; Mestres, C.; Busquets, M. A. J. Colloid Interface Sci. 1999, 211, 130.

Sospedra et al.

general trend observed from these plots indicates that the interaction with lipids decreases in the order A3 > VP3 . A2. The charge of the lipid monolayer also plays a role on surface pressure increase. A3 is neutral and has a high activity in negative monolayers if compared to VP3 and A2, both negatively charged. This fact indicates a predominance of hydrophobic interactions over electrostatic ones for A3 and also that electrostatic interactions are negatively affecting the interaction of A2 and VP3 with negatively charged surfaces. Activity of VP3 and A3 on SM monolayers is very similar although we can predict a different mechanism, precisely related to the charge of both lipid and peptides. SA has a positive charge, which explains the high affinity showed for VP3 thus indicating electrostatic interactions. In the case of A3, interaction is as before, related to the hydrophobic acyl chain of SA. What is surprising are the results obtained for A2 and VP3. Both peptides have a net negative charge, but compared to VP3, A2 has a very low affinity for the monolayers, specially for those of negative charge. There is also a distinction in activity among negatively charged phospholipids and the A2 and VP3. A2 has little activity on PS monolayers at the initial pressure (π0) of 5 mN/m and it disappears completely at the π0 of 10 and 20 mN/m. This point can be explained for A2 in this model as a predominance of electrostatic interactions over hydrophobic ones maybe due to differences on conformation adopted at the air/water interface while for VP3 there is a balance between hydrophobic and electrostatic forces. Previous studies performed with VP3 by means of compression isotherms,21 in which the change of surface pressure (π) versus area (A) occupied by molecule was analyzed, showed that interaction of the peptide with neutral and positively charged lipids was mainly hydrophobic, while when the lipid was negative there was a predominance of electrostatic forces. In addition, an expanding effect on monolayers was independent of the electrical charge of the lipids.22 More evidence about the combination of hydrophobic and electrostatic interactions was observed for VP3 by means of circular dichroism studies. In this case, VP3 showed a preferably ordered β-structure in negatively lipid vesicles composed of PC/ PG,4 in contrast with the results observed with PC (zwitterionic) and PC/SA (positively charged) vesicles in which the peptide presented a random coil structure. Compression Isotherms. The three peptides were able to form stable monolayers that gave on compression regular isotherms. Area/residue values were consistent with stable monolayers, and dissolution in the subphase could be discarded. As confirmed by compression and decompression cycles, parts a and b of Figure 3 show the isotherms (π versus A) obtained when compressing pure films of A2 and A3, respectively, spread on a PBS (pH 7.4) interface. As observed for the parent compound, VP3 (figure not shown),21 A2 and A3 isotherms show the presence of the expanded and the liquid-condensed phases as the film is compressed and a low collapse pressure about 30 and 22 mN/m, respectively. These reported surface areas were expected as described in the literature for peptides of similar length.23-25 However, VP3 and A2 isotherms are about 10-fold more expanded if compared (21) Sospedra, P.; Alsina, M. A.; Espina, M.; Reig, F.; Haro, I.; Mestres, C. J. Colloid Interface Sci. 2000, 221, 230. (22) Mestres, C.; Pe´rez, J. A.; Reig, F.; Haro, I.; Alsina, M. A. Colloid Polym. Sci. 1997, 275, 946. (23) Maget-Dana, R. Biochim. Biophys. Acta 1999, 1462, 109. (24) Vie´, V.; van Mau, N.; Chaloin, L.; Lesniewska, E.; Le Grimellec, C.; Biophys. J. 2000, 78, 846. (25) Dieudonne´, D.; Mendelsohn, R.; Farid, R. S.; Flach, C. R. Biochim. Biophys Acta 2001, 1511, 99.

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Figure 4. Peptides orientation in an air/water interface based on the Chou and Fasman conformational model.

to that of A3. Usually, monolayers are in a more expanded state when the components are charged, due to repulsive interactions.26 In our case, these results could be explained in terms of conformation differences at the air/water interface adopted for the peptides. From the Chou and Fasman conformational studies,10 one could expect a β-turn for A3 in the amino acid region RKDL as indicated in Figure 4. Although A3 has a net neutral charge, it has both a positive and a negative charge due to K and D amino acids, respectively. The charges are more exposed to the air/water interface while the rest of the peptide chain, which is more hydrophobic, would be located out of the water. This would also explain the higher penetration of A3 into lipid interfaces if compared with VP3 and A2. DSC Studies. Lipid membranes are characterized by a thermotropic phase transition between an ordered gel state and a disordered liquid-crystalline phase. Bilayer modification is accompanied by several structural changes in the lipid molecules. The principal change associated to such transition is the trans-gauche isomerization in the acyl chains. The average number of gauche conformers can be related to the bilayer fluidity.27 In addition, the presence of a drug molecule susceptible of interdigitate among the phospholipid molecules causes perturbations that contribute to the bilayer fluidity. In the present paper, the thermotropic phase behavior of zwitterionic DPPC MLVs in Tris buffer (pH 7.4) was studied by DSC, in the absence and in the presence of increasing concentrations of VP3, A2, or A3. DPPC pure bilayers display two endothermic transitions: a gel to gel (Lβ′-Pβ′) pretran-

sition, corresponding to the melting point of the lipid headgroups, centered at 35.13 ( 0.07 °C, and a main transition at 41.17 ( 0.07 °C that corresponds to the highly cooperative gel to liquid-crystalline transition (Pβ′-LR) (chain melting) of the lipid side chains. The entalphy, ∆H, of the gel/liquid-crystalline transition of DPPC found in our experiments equals 34.6 ( 1.4 kJ/mol, in good agreement with the literature.28 The effects of the peptides on the thermotropic phase transition properties of DPPC MLVs were investigated by DSC at the mixtures containing 0, 10, 20, 30, or 40 mol % of peptide. At these compositions none of the peptides clarified the turbidity due to DPPC vesicles. The heating endotherms corresponding to the second heating of the DPPC vesicles alone or with VP3, A2, or A3 peptides are shown in Figure 5. As expected, DPPC pretransition is abolished in all peptide-containing samples. This is a common effect observed with drugs of different characteristics and does not provide any information about the location of the peptide in the lipid bilayer. Although the peptides do not show a significant displacement of the phase transition midpoint of the DPPC (Table 3), the broadening of the transition profile of DPPC bilayers with increasing amount of peptides is considerable. A 10 mol % portion of peptide added to the MLVs produces about 50% peak reduction, and it disappears at above 20 mol % for all peptides. This difference could be attributed to the net charge of the peptides. VP3 and A2 are anionic peptides, while A3 is neutral and can probably better insert into the lipid bilayers. Main transition broadening, described as peak width at half-height (∆T1/2), means that the size of the cooperative undergoing transition decreases with peptide concentration and that there is also an expansion on the temperature range of density fluctuations, resulting from dynamic lateral bilayer heterogeneicity, in terms of formation of lipid domains and associated interfacial regions.29 The higher the percentage of peptide into the bilayer, the greater the broadening of the transition (Table 2). ∆H also decreases with peptide content. This decrease in ∆H values suggests that increasing amounts of peptide in DPPC not only perturbed the bilayers but also significantly reduced the intermolecular interactions between bilayer interiors, probably caused by the disruption of hydrogen bonding at the lipid-

(26) Capuzzi, G.; Lo Nostro, P.; Kulkarni, K.; Fernandez, J. E. Langmuir 1996, 12, 397. (27) Nerdal, W.; Gundersen, S. A.; Thorsen, V.; Høiland, H.; Holmsen, H. Biochim. Biophys. Acta 2000, 1464, 165.

(28) Videira, R. A.; Antunes-Madeira, M. C.; Madeira, V. M. C. Biochim. Biophys. Acta 1999, 1419, 151. (29) Mouritsen, O. G.; Jøregensen, K. Chem. Phys. Lipids 1994, 73, 3.

Figure 3. Pressure-area isotherms for (a) A2 and (b) A3 peptides spread at the interface on a PBS buffer subphase (pH 7.4).

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Figure 5. DSC heating endotherms of DPPC (4 mM) MLVs prepared in the presence of (O) 0, (b) 10, (0) 20, (9) 30, and (4) 40 mol % of (a) VP3, (b) A2, and (c) A3. The curves refer to the second scan in the heating mode at a temperature scanning rate of 5 °C/min. Table 3. Thermotropic Parameters of the Gel to Liquid Crystalline Phase Transition of DPPC MLVs Prepared in the Presence of Different mol % of VP3, A2, and A3 Peptides DPPC VP3 A2 A3

10% 20% 10% 20% 10% 20%

Tma (°C)

∆H (kJ/mol)

∆T1/2b (°C)

41.17 41.20 41.36 41.03 41.12 41.24 41.27

35.35 27.50 16.11 23.30 21.60 22.18 18.90

0.66 1.05 1.75 0.99 1.22 0.89 1.08

a Main transition peak temperature. b Temperature width at half-height of the heat absorption peak.

water interface.30 It should be noted that the shape of the thermograms and the ∆H values obtained from the second temperature scan of the same sample do not change, (30) Ali, S.; Minchey, S.; Janoff, A.; Mayhew, E. Biophys. J. 2000, 78, 246.

suggesting that the phase transition of DPPC is reversible at all peptide/PL ratios. A broadening of the main transition without any change in the peak melting temperature is characteristic of molecules localized in the outer cooperative zone of the bilayer. Therefore, these calorimetric findings are compatible with an interfacial location of the peptides and with some penetration of the nonpolar amino acid side chains into the hydrocarbon chain region closer to the polar/non polar interface. Fluorescence Studies. Taking into consideration that the emission spectra of Trp is sensitive to its environment and that VP3, A2, and A3 have Trp residues, we have monitored peptide interaction with lipid vesicles by fluorescence spectroscopy. At a excitation wavelength of 285 nm, the electromagnetic radiation is almost entirely absorbed by Trp and the corresponding emission spectra yield information regarding the surroundings of Trp residues in peptide sequences. The emission maximum of buried Trp residues ranges between 326 and 338 nm, and the corresponding maximum for exposed Trp is about 350356 nm. As a membrane model system for this study, we choose small unilamellar liposomes (SUVs) composed of the zwitterionic DPPC and a mixture of DPPC/DPPG (95/5) with a negative charge to analyze the magnitude of electrostatic interactions. In addition, experiments were performed at room temperature and at 50 °C, under and above, respectively, the main transition temperature (Tm) from gel to liquid crystalline state of DPPC to study the effect of membrane fluidity on lipid/peptide interaction. Parts a and b of Figure 6 show the changes in Trp wavelength emission maximum (λmax) for VP3, A2, and A3 alone and in the presence of SUVs at room temperature and at 50 °C, respectively. At room temperature, peptides had a λmax (average of three determinations) of 353 nm, indicating a polar environment for the Trp residues. In the presence of DPPC vesicles, VP3 and A3 show a blue shift with λmax values of 344 and 340 nm, respectively. This value is not clearly indicative of a buried location for Trp, and we could assume a combination of the two orientations into the liposome bilayer. Under the same analytical conditions, A2 shows a slight blue shift with a λmax of 350 nm indicating no change on Trp orientation with respect to the free peptide in solution. On another hand, liposome charge does not seem to play any role on VP3 and A2 because λmax has the same value independently of the lipid composition. Consistent with these results, previous fluorescence studies4 carried out with VP3 and liposomes revealed that the interaction of the peptide in the gel state did not change in the presence of negatively charged phospholipids. Contrarily, A3 shows clearly an exposed environment for Trp in the presence of a 5% of DPPG indicating a low degree of penetration into the bilayer. Maximum emission for free peptides at 50 °C has a notable blue shift. As far as the influence of the fluidity on the membrane in lipid/peptide interaction is concerned (Figure 6b), above DPPC Tm, there is a red shift for the peptides (338-346 nm) in the presence of SUVs if compared to the peptides in buffer solution (335 nm). For DPPC/DPPG (95/5) liposomes, the trend is similar to the observed at room temperature, being the values of λmax for VP3, A2, and A3 of 338, 342, and 346 nm respectively. While free peptide in solution has the Trp clearly buried, maybe due to the peptide aggregation as previously

Peptide-Membrane Interaction

Langmuir, Vol. 18, No. 4, 2002 1237

range from 5 to 35 °C. When the same peptides were incubated with liposomes, only VP3 showed an ordered β structure over the same temperature range. In the same set of experiments, and to discard the formation of a β-sheet due to peptide aggregation, the ability of VP3 to interact with liposomes was assayed as a function of peptide concentration. The results obtained indicated the absence of intermolecular aggregation, since the more diluted the samples were, the higher the β structure observed, thus suggesting the structure to be a β turn.4

Figure 6. Variation of tryptophan wavelength emission maximum (λmax) for VP3, A2, and A3 in the presence of phospholipid vesicles at (a) room temperature and (b) 50 °C.

observed for the longer peptide sequence VP3 (102-121),31 it remains unclear what is the orientation of the peptide in the lipid monolayer. By this technique it is difficult to elucidate if the observed λmax decrease in the presence of liposomes is due to a real interaction of the peptides with phospholipids. However, if compared with results obtained by DSC, we can assume a preferred interaction of peptides with liquid crystalline state over the gel state. That means that the interactions above Tm are predominantly hydrophobic. Increments obtained in the fluorescence intensity after scattering and inner filter corrections were consistent with the above-reported results. Previous CD studies performed4 to analyze temperature effect on peptide conformation indicated that peptides in solution showed a random coil structure in a temperature (31) Garcı´a, M.; Pujol, M.; Reig, F.; Alsina, M. A.; Haro, I. Analyst 1996, 121, 1583.

4. Conclusions We have used several techniques to analyze the influence of the change in amino acid sequence on peptide physicochemical properties. Our results suggest the following features: (1) VP3, A2, and A3 are able to form stable monolayers when spread at an air/water interface, A2 isotherms being more expanded that those of VP3 and A3, thus indicating that not only the charge of the peptide but also the conformation of the peptide at the air/water interface has to be considered. (2) Substitution of Asp (D) amino acid (VP3) for Glu (E) (A2), both giving a net negative charge to the peptide, results in a change in the type of interaction with monoand bilayers. ∆π of A2 with negative lipid monolayers is very low at 5mN/m and disappears at higher initial pressures, thus indicating a repulsion of the polar heads and, consequently, a predominance of electrostatic interactions. However, results obtained with the same peptide and neutral phospholipids showed higher ∆π values, a fact indicative of a hydrophobic interaction. In conclusion, interaction depends on the phospholipid nature. (3) Substitution of Gly (G) (VP3) for Lys (K) (A3) does not change significantly the physicochemical properties of the peptides. VP3 and A3 follow a similar trend in all the applied techniques despite having different charge (negative and neutral, respectively). A3 shows higher interaction with lipid monolayers regardless of the charge, a fact indicative of a predominance of hydrophobic interactions. VP3 shows higher interaction with neutral than with negatively charged phospholipids coexisting a balance of hydrophobic and hydrophilic forces. (4) The binding of the peptides to lipids results in a decrease in the entalphy of the heat-induced phase transition of zwitterionic vesicles at low peptide concentration, independently of the peptide charge. (5) Fluorescence and DSC studies show a preferred interaction of peptides with liquid crystalline state over the gel state. This report represents one more step toward establishing a thermodynamic database needed to gain insights into the molecular forces that govern the binding of HAV proteins to lipid membranes. Acknowledgment. We thank J. Carilla Auguet and A. Lo´pez Navarro (Laboratori d’Ana`lisi Te`rmica IIQAB, CSIC, Barcelona) for excellent technical assistance in DSC measurements. This work was supported by Grant BIO 95-0061-C03-03 from CICYT (Spain) and a predoctoral CIRIT grant (1997 FI000073) awarded to P. Sospedra. LA011156V