Langmuir 1993,9, 1129-1133
1129
Miscibility of HBV Peptides and Dipalmitoylphosphatidylcholine in Monolayers M. A. Alsina,*tfC. Meshes,+F. Rabanal,t M. A. Busquets,?and F. Reigt Unitat de Ftsico-Qutmica, Departament de Farmbcia, Facultat de Farmbcia, Universitat de Barcelona, Avda Joan XXIII, sln 08028 Barcelona, Spain, and Departament de P&ptids, CID-CSIC, Jordi Girona Salgado 18-26, 08034 Barcelona, Spain Received April 22,1992. In Final Form: January 19, 1993 Three lipopeptidesderived from HBV-S(139-148)sequence containingstearoyl, cholanoyl, and Pam3Cys(Ser)zmoieties were studied as far as their interactions with phospholipids are concerned. The parent compound and the three analogues have surface activity and penetrate lipid monolayers composed of DPPC. The miscibility of these peptides with the same phospholipid was nearly ideal. The area molecule values calculated for the parent peptide suggest an a-helicalstructureand the predicted secondarystructure for this sequence,determined by the Chou and Fasman parameters,is also consistentwith this conformation. The lipophilic derivatives show, nevertheless,higher molecular areas that fit better with an a-helix and 8-sheet segments linked by a 8-turn. The Pam3Cys(Ser)zderivative showed an anomalous behavior both in HPLC and in monolayer experiments, probably the bulkiness of the hydrophobic moiety gives preferentially a micellar structure.
Introduction It is now generally accepted that small peptide fragmenta of viral proteins, usually coupled to a large protein carrier, may elicit an antibody response in experimental animals, thus offering the possibility of preparing a new generation of vaccines.' Nevertheless, the physicochemical basis of the interactions involved in the recognition and antibody generation processes remain to be clarified. It is difficult to explain that the cognate protein would be able to recognize an antibody raised against a small peptide lacking in general of secondary structure, unless, one accept that the interaction between the peptide and the carrier would promote some kind of structuration on the peptide backbone. In this sense Dyson et aL2 have suggested that the most highly preferred conformation of the free peptide in water is stabilized on the surface of the carrier or becomes stabilized when the peptide binds to the B cell receptor. Liposomes have been recently used as immunoadjuvants, either containing entrapped peptides or having them linked to their surfacem3v4 The mutual interactions between biologically active peptides and lipids have been the subject of study by different g r ~ u p s . ~The . ~ accumulated knowledge in this field shows that lipids act to stabilize the preferred ordered structures of peptides. This situation could be similar to the interactions found when the peptide comes first in contact with the B cell receptor. For this reason, membrane models are a convenient tool to determine the most populated conformations of potentially antigenic peptides. The S viral protein of hepatitis B has been shown to contain severalwell-definedepitopes. One of them, located in the fragment 139-148, has been recently synthesized in our laboratory, along with three hydrophobic derivatives.7 These four peptides share in common the peptide back-
* To whom the correspondence should be addressed.
(1)Leoner, R. A. Adv. Immunol.1984, 36. (2)Dyson, H.J.; Lerner, A. R.; Wright, P. E. Annu. Rev. Biophys. 1988, 17, 305. (3)Garcon, M. J. N.; Six, R. H.J. Immunol. 1991 146 (ll),3697. (4) Frisch, B.;Muller, S.;Briend, J. P.; van Regenmortel, M. H.V.; Schuber, F. Eur. J.Immunol.1991,21, 185. (5)Gob, Y.;Hagihara, Y. Biochemistry 1992,31, 732. (6)McLean, L. R.; Hagaman, K. A.; Owen, T. J.; Krestenansky, J. L. Biochemistry 1991, 30, 31.
bone, but three of them have a hydrophobic residue linked to the amino terminal: cholanoyl, stearoyl, and Pam3Cys-Ser-Seryl. In this paper we describe the surface activity of these peptides are their miscibility pattern with phmphatidylcholine, one of the major constituents of liposomes and cellular membranes, using monomolecular layers as a membrane model. The study of miscibility can be approached in two ways, either determining changes in the phase transition, as function of monolayer composition, or calculating the area/ molecule values of mixed monolayers. None of these processes is devoid of inaccuracy, for this reason we have applied both criteria to study the interactions of our peptides with DPPC. This phospholipid was chosen due to the presence of a clear phase transition around 7-8 mN1m.
Materials and Methods Chemicals. The synthesis of the peptides whose structures are given in Figure 1 is described elsewhere.* DPPC was purchased from Sigma and its purity, checked by thin-layer chromatography, was shown to be higher than 99%. Chloroformand methanol (pro-analysi)were from Merck. Water was double distilled in an all-glass apparatus. Compression Isotherms. The experiments were performed on a Langmuir film balance equipped with a Wilhelmy platinum plate, following the procedure given in ref 9. The Teflon trough (495cmZ,330 cm3) was regularly cleaned with hot chromic acid as well as the platinum plate. Films were spread on the aqueous surface (PBS, pH 7.4),with a Hamilton microsyringe, and at least 10 min was allowed for solvent evaporation. All the isotherms were run at least 3 times in the direction of increasing pressure with freshly prepared films. The measurements were made at 21 f 1 O C . The samples were dissolved in a mixture of chloroform/ methanol (60/40 (v/v))and different volumes of peptides and phospholipid solutionswere mixed in order to achievethe desired molar compositions. (7)Rabanal, F.; Haro, I.; Reig, F.; Garcia Antbn, J. M.J. Chem. SOC. Trans. 1991, 1, 945-951. (8)Rabanal, F.; Haro,I.; Reig, F.; Garcfa Antbn, J. M. Znt. J . Peptide Protein Res. 1990, 36, 26. (9)Alsina, M. A,; Mestres, C.; Garcfa-Ant6n, J. M.; Espina, M.; Ham, I.; Reig, F.Langmuir 1991, 7 (5),975-977.
0743-746319312409-1129$04.00/0 0 1993 American Chemical Society
Alsina et al.
1130 Langmuir, Vol. 9, No. 4, 1993 a) [Tyr148JS(i3s.14s): 139Cys-Thr-Lys-Pro-Thr-A~p-Gly-Asn-Cys-Tyr~ b)
Table I. Surface Pressures Corresponding to the Phase Transition of DPPC/S-(139-148) Mixed Monolayers at Different Molar Compositions molar fraction molar fraction DPPC/peptide ll (mN/m) DPPCipeptide Il (mN/m) 1/0 7.83 0.6/0.4 8.64 0.810.2 8.64 0.4/0.6 9.45 Table 11. Compressibility of DPPC/S-( 139-148) ,Mixed Monolayers Measured at 5 and 15 mN/m compressibility (m/mN) molar fraction DPPC/peptide 5 mN/m 15 mN/m 1/0 2.44 X IO-' 0.80 X 0.8/0.2 2.50 X 10-2 0.99 x IO-' 0.6/0.4 2.01 x 10-2 0.84X lo-' 0.410.6 2.05 X 0.98 X 0.210.8 1.78 X lo-* 1.10x 10-2 Oi 1 1.15 X 0.50 X
".cwoI *'cw* w
.
c
i
0
0
Figure 1. Chemicalstructuresof thepeptides: (a)parent peptide sequence; (b) cholanic acid; (c) PamnCys-Ser-Serresidue.
Moreover, there is a dramatic change in the isotherm compressibility after the phase transition, thus indicating a change in the orientation of the peptide in the monolayer. The figures corresponding to the compressibility at 5 and 15 mN/m calculated according to eq 1 (where A is the molecular area in the film at a fixed pressure), are given in Table 11.
The inset in Figure 2 represents the mean areas of the mixtures calculated at different superficial pressures. There are small deviations from ideality, reflecting the predominance of either a lack of interactions or an ideal miscibility. To better quantify the miscibility characteristics of these two Components in the monolayer, the thermodynamicparameters associated to this process have been calculated. To this end, the excess free energy of mixing and the interaction parameters were calculated by applying the following equation
(2)
Figure 2. Compression isotherms of mixed monolayers of DPPC/S (139-148) peptide spread on PBS subphases (pH 7.4). Molar fraction of DPPC: 0 , O ; A, 0.2; *, 0.4; Q0.6;m, 0.8;0 , 1. Inset meanarea/moleculein mixed monolayersof DPPC/S (139148) peptide. Determinations were made at different surface pressures (mN/m): D, 5, 0,10,m, 15; and X, 20.
Results and Discussion Surface Activity Studies. The miscibility pattern of lipid-peptide samples and the area-molecule values calculated from the isotherm registers are a valuable tool to predict liposomal stability and peptide preferred conformations in this hydrophobic media. Pure DPPC compression isotherms exhibit a phase transition around 8mN/m. Mixed monolayers of this lipid with the S-peptide show the same type of transition but the phase change appears at slightly higher compression pressures as the content of peptide in the mixture increases (Figure 2). The above cited values are given in Table I.
where AI, is the mean molar area in the mixed film, A1 and A2 are the molar areas of the two pure components, and N1 and NZ are the molar fractions of monolayer components 1 and 2 as was already described by Alsina et aL9 The values of the interaction parameter (a)at different pressures have been calculated by applying eq 3 derived from Joos et al.10-12 and Margules13
ACME' (3) RT{x,x,' + xZxl2) x1 and xp are molar fractions (1 and 2 being the same components as defined for eq 2). These parameters are summarized in Table 111. One can appreciate that in all cases AG is lower than RT (2474.56 J/mol at 25 "C)thus confirming the low interaction level detected. The area/molecule of the peptide at 20 mN/m is 0.33 nm2. This is an extremely low value suggesting the a=
(10) Joos, P. Bull. SOC.Belg. 1969, 78, 207. (11) Joos, P.; Demel, R. A. Biochim. Biophys. Acta 1969, 283, 447. (12)Joos, P.; Ruyssen, R.; Mifiones, J.; GarcIa Fernhdez, S.;Sanz Pedrero, P. J . Chim. Phys. Physicochim. Biol. 1969,66 (lo), 1665.
(13)Classtone, S. In Thermodynamics for Chemists; Ed Aguilar (Spanish version), 1972;Chapter XIV.
Miscibility of Lipopeptides in Monolayers
Langmuir, Vol. 9, No. 4, 1993 1131
Table 111. Excess Free Energy of Mixing ( A G E x ~J/mol) , and Interaction Parameters (a)in Mixed Monolayers of DPPC/S-( 139-148) molar fraction DPPC/peptide 0.8/0.2 0.610.4 0.4/0.6 0.2/0.8
10 mN/m AGEx~* a 278 0.71 102 0.17 0 0 253 0.65 WI\CIllILIrV
20 mN/m a
524 174 -37 452
1.34 0.30
0 1.16
Table V. Excess Free Energy of Mixing ( A G X ~J/mol), , Interaction Parameters (a),and Energies (AH, J/mol) in Mixed Monolayers of DPPCKholanoyl-5-( 139-148) 10 mN/m 20 mN/m molar fraction A G E x ~ * AH AG"# AH DPPC/peptide (J/mol) a (J/mol) (J/mol) a (J/mol) 0.8/0.2 273 0.70 855 561 1.43 877 -3893 -2.37 -2897 -1034 -1.76 -1077 0.6/0.4 -198 -0.34 -413 -259 -0.44 -270 0.410.6 463 1.19 1449 1006 0.210.8 2.57 3145 ~~
D F F l ( I I O I A h O l l \ (119 1 4 x 1
the transition from expanded to condensed liquid is soft and differences in compressibility are very low. The molecular area at 30 mN/m is 0.87nm2,this value can be associated to the presence of an expanded @ sheet structure stacked perpendicular to the interface. The inset in Figure 3 shows the mean area/molecule as a function of the monolayer composition, There is a slight compression of the monolayer but the energies associated to this variation are, as in the preceding case, very low. In these series of isotherms, as monolayers collapse, the interaction energies could be calculated by applying eq 4 (Table V).
lb
12
2b
Area (nma molcc-')
Figure 3. Compression isotherms of mixed monolayers of DPPC/ cholanoyl-S (139-148) peptide spread on water subphases (pH 7.4). Molar fraction of DPPC: 0,0; A, 0.2; *, 0.4;0,0.6; W, 0.8; 0,l. Inset: mean area/molecule in mixed monolayers of DPPC/ cholanoyl-S (139-148) peptide. Determinations were made at different surface pressures (mN/m): 0,5; 0, 10; B, 15; X, 20. Table IV. Surface Pressures Corresponding to the Phase Transition of DPPC/Cholanoyl-S-(139-148) Mixed Monolayers at Different Molar Compositions molar fraction II transition molar fraction ll transition (mN/m) DPPUpeptide (mN/m) DPPC/peptide 0.4/0.6 32.16 1/0 7.83 40.32 0.2/0.8 0.8/0.2 28.80 0.6/0.4 o/ 1 31.20 40.80
possibility of a partial solubilization process. According to Fidelio,14the minimal surface area for a @ sheet is 0.7 nm2, but in a recent paper Jung et al.15 found 0.54 nm2/ molecule for a short PamaCSS peptide. Considering these data and the sequence and length of our peptide, the values we found could correspond to a highly packed stable monolayer. Hydrophobic Peptide Derivatives. Mixed monolayers of DPPC/Cholanoyl S peptide (139-148)show, on compression,isotherms (Figure 3)containing two phases: expanded and condensed liquid. Moreover, in this set of experiments a clear collapse point can be detected at any molar composition. The phase transition appears at different surface pressures depending on the composition of the monolayer, being more evident at DPPC/peptide 0.2/0.8. These values are given in Table IV. Nevertheless, (14)
Fidelio, C. D.; Austen, B. M.; Chapman, D.; Lucy, J. A. Biochem.
J. 1996,238, 301.
(15) Prass, W.; Ringsdof, H.; Bessleer, W.; Wiesmuller, K. H.; Jung, G . , Biochim. Biophys. Acta 1987,900, 116.
Ah = RTa/Z (4) For the determination of the coordination number (Z), we followed the model of Quikenden and Tam16considering that in a closely packed monolayer (collapse), each molecule is surrounded by six neighbors. For lower pressures (10 and 20 mN/m) eq 5 was applied to calculate the packing fraction (PF). This value was used to obtain the corresponding 2,according to que equivalences given in ref 16, where A,, is the area-molecule of the mixture at the collapse point and A , the area-molecule of the mixture at 10 or 20 mN/m. Considering the calculated values for the energies, it is clear that miscibility is nearly ideal being in no case higher than RT. P F = 0.907AmC/A,
(5)
Nevertheless, in some cases it is possible for films at low pressure to be miscible with phase separation occurring on compresion,but before collapse. When the film collapse is easily detectable, measuring the collapse pressure over the range of film compositions can give information as to whether a true mixed film exists. When both components are miscible collapse pressure will vary with composition and the following equation can be applied
were x1 and x2 are mole fractions of components 1 and 2 in the monolayer, nc,land nc,2are collapse pressures of the single components 1and 2,nc,m is the collapse pressure of the mixed film, and 01 and 0 2 are molar areas of component 1 and 2 at the collapse point. After substitution of experimental values to the above equation we found that the sum of both exponentials is a little bit lower than 1, ranging from 0.80 to 0.97. This is indicative that the miscibility is nearly ideal. However, the fact that the surface transition does not disappear at increasing content of the other component may indicate that the mixing is incomplete. Stearoyl-S-Peptide. Monolayers of this peptide correspond to a liquid expanded at low pressures and liquid condensed at higher pressures (Figure 4). The isotherms (16)Quickenden, T. I.; Tan, C. K. J. Colloid Interface Sci. 1974,48
(3), 382.
Alsina et al.
1132 Langmuir, Vol. 9, No.4, 1993
Area (nml molcc-I)
Figure 4. Compression isothermsof mixed monolayers of DPPC/ stearoyl-S(139-148) peptide spread on water subphases(pH 7.4). - - -,0.6;., 0.8; Molar fraction of DPPC: - - -, 0; &0.2;-,0.4; .,I. Inset: mean area/molecule in mixed monolayers of DPPC/ stearoyl-S (139-148) peptide. Determinations were made at different surface pressures (mN/m): 0,5; 0,10; B, 15; X, 20. Table VI. Excess Free Energy of Mixing ( A G l m , J/mol), Interaction Parameters (a),and Energies (AH, J/mol) in Mixed Monolayers of DPPC/Stearoyl-S-( 139-148) 10 mN/m 20 mN/m molar fraction A C E x ~ * AH AGExM* AH DPPC/peptide (J/mol) a (J/mol) (J/mol) a (J/mol) 1579 3.40 1076 656 1.68 1025 0.8/0.2 648 1424 2.43 1483 622 1.06 0.6/0.4 0.4/0.6 473 0.81 493 907 1.55 944 -278 -0.71 -435 0.2/0.8 -64 -0.16 -100
achieve the collapse points without reaching a solid state. The inset shows the miscibility of peptideDPPC. It is clear that the process is nearly ideal at any surface pressure. The area-moleculeoccupied by the pure peptide measured at 30 mN/m was 1.11 nm2. This value is intermediate between an a-helix (1.6 nm2) and @-sheet(0.7 nm2) and could correspond to the presence of a @ turn. According to the predicted structures by Chou and Fasmann parameters, these peptides have no clear @-sheetor a-helix structures. For this reason, the most likely secondary structure of this peptide derivative in a very rigid matrix is a 8-sheet with the carboxyl and phenolic hydroxyl of the tyrosine in contact with the aqueous subphase and the rest of the peptide perpendicular to the interface, having also a @ turn at the N terminal amino acid. The mixing excess energies, interaction parameters, and interaction energies were calculated for all the mixtures at 10 and 20 mN/m as described before. All these values are given in Table VI. As stated for the cholanoyl derivative, the energies associated to the mixing process are very low, thus pointing toward a nearly ideal behavior. Moreover, as in the cholanoyl derivative, eq 6 was applied to the collapse values found for mixed monolayers and the s u m of both terms of the equation is lower than 1,ranging from 0.81 to 0.93, thus confirming small deviations from ideality determined from the interactions energies.
Area (nm2 molec-'J
Figure5. Compressionisotherms of mixed monolayers of DPPC/ Pam3-Cys-Ser-Ser-S(139-148) peptide spread on water subphases (pH 7.4). Molar fraction of DPPC: 0,0; - - -, 0.25; - - -,0.5; -, 0.75; .,I. Inset: mean area/molecule in mixed monolayers of DPPC/Pam3-Cys-Ser-Ser-S(139-148) peptide. Determinations were made at different surface pressures (mN/m): D,5; 0, 10; B, 15; X, 20. Table VII. Compressibility of DPPC/ Pam-Cys-Ser-Ser-5-(139-148) Mixed Monolayers Measured at 5 and 15 mN/m molar fraction DPPC/peptide *I I 1/0 0.25/0.75 05/05 0.25/0.75 0/1 1.7 X 1k2 1.6 X 1k2 5 1.2 X 10-2 1.9 X 1k2 2.1 X 15 0.9 X 10-2 2.1 X lo-* 3.3 X le2 1.9 X 1k2 1.4 X 1k2 ~
~
~~
Table VIII. Excess Free Energy of Mixing ( A P M , J/mol) and Interaction Parameters (a)in Mixed Monolayers of DPPCICys-Ser-Sed-(139-148) molar fraction DPPC/peptide 0.25/0.25 0.5/0.5 0.2510.75
10 mN/m AGEx~* a 2341 5.11 3316 5.42 1960 4.22
20 mN/m AGE%* a 3899 8.50 5199 8.50 3141 6.85
PamaCys-&Peptide. Isotherms of pure peptide (Figure 5) showed, at very low surface pressures, a liquid expanded state, from 2-3 to 20 mN/m the isotherm corresponds to a liquid condensed state; at this point there is a clear phase change to a solid state. Due to the striking differences in the shape of isotherms before and after the phase change, the compressibilities of all of them were calculated at 5 and 15 mN/m and are given in Table VII. Mixed monolayers gave also the same phase change, but when increasing the amount of DPPC there is a tendency toward lower surface pressures. The isotherms of pure components and their mixtures are given in Figure 5 and in the inset the area-molecule of the mixed monolayers are represented as a function of the molar composition. At any pressure one can appreciate that there is an expansion of the monolayer, this effect being more evident at lower surface pressures. The molecular area at 30 mN/m calculated for the pure peptide is 1.8nm2/molecule. According to the values found
Miscibility of Lipopeptides in Monolayers by Jung15et al. of 0.5 nm2for the minimal area of a trialkyl hydrocarbon chain, the value found in our determinations could be due to the existence of a @-turnbetween the peptide and the tripalmitoyl derivative. The thermodynamicparameters of thesemixtures were calculated by applying the equations described before and the values are given in Table VIII. In this case it was not possible to calculate the interaction energies due to the lack of a real collapse point. As energies are in all cases positive, this means that mixed monolayers are less stable than pure components.
Langmuir, Vol. 9, No. 4, 1993 1133 There is a predominance of repulsion forces among lipid and peptide molecules. This behavior is not easy to understand due to the similarity between the phospholipid derivative and the phospholipid moiety of the peptide; one should expect an ideal miscibility.
Acknowledgment. This work was supported by a grant, Far-884692, from CICYT, and a CEE project, BAP0469-E (IR). We wish to thank Marla de la Sierra Oauna for technical assistance.
