Free-Standing Black Films: An Alternative to Langmuir Monolayers for

Dec 18, 2001 - This paper reports on a study by Raman spectroscopy of free-standing black films of dimyristoylphosphatidylcholine (DMPC) in interactio...
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Langmuir 2002, 18, 512-518

Free-Standing Black Films: An Alternative to Langmuir Monolayers for the Study by Raman Spectroscopy of Peptide-Phospholipid Interaction in Ultrathin Films F. Lhert, D. Blaudez,* C. Heywang, and J.-M. Turlet Centre de Physique Mole´ culaire Optique et Hertzienne, UMR 5798 du CNRS, Universite´ Bordeaux I - 351 cours de la Libe´ ration - 33405 Talence, France Received August 6, 2001. In Final Form: October 1, 2001 This paper reports on a study by Raman spectroscopy of free-standing black films of dimyristoylphosphatidylcholine (DMPC) in interaction with melittin, a hemolytic peptide. In a first step, the Raman spectra of these films are compared with those of Langmuir monolayers, using the same acquisition conditions. Black films give much better spectra than Langmuir monolayers and, hence, are used to follow the effects induced by melittin at different melittin/DMPC molar ratios ranging from 1/1000 to 1/55. Two parameters deduced from the intensity of some specific bands in the Raman spectra show that melittin induces an increase of the lateral and conformational order in DMPC alkyl chains. Moreover, increasing amounts of melittin induces an increase of the water core thickness of black films from 22 to 400 Å. This swelling is explained in terms of the DLVO theory. For the thinnest films, the shape of the multicomponent water band changes which reveals a confinement effect in the aqueous core. Finally, discussion of these results is oriented toward the suitability of the black film configuration for Raman studies of peptidephospholipid interaction in ultrathin films.

Introduction Understanding the interaction of proteins with membranes is of fundamental interest, since this interaction is a decisive factor for biological activity and, in some cases, induces by itself strong effects such as lysis or fusion. Beside studies done directly on living cells, it is valuable to use simple models of interfaces where subtle changes in molecular organization can be more easily observed. In this context, Langmuir lipid films spread at the air-water interface have been widely used since they are easy to set up, they allow wide variation of the molecular packing, and they enable study of the effect of biologically active molecules.1 Raman spectroscopy could be a very convenient experimental approach to follow protein-lipid interactions at interfaces. Spectra provide a direct molecular information and are very sensitive to changes in lateral and conformational order of lipid alkyl chains. With the help of cooled multichannel CCD detectors, Raman spectroscopy has gained enough sensitivity to detect monolayer signals.2,3 However, in the case of Langmuir monolayers, and more generally with nanosamples immersed or in contact with a deep aqueous subphase (vesicle solutions, black lipid membranes, ...), Raman spectroscopy suffers from the very large difference in intensity which exists between the very weak signal originating from the monolayer (about one count per second for the CH stretching bands) and the very strong contribution of the water subphase (about 103 counts/s for the OH stretching bands). Confocal microscopy configuration will limit this subphase contribution to the first few micrometers, but it does not help too much. * To whom correspondence should be addressed. Fax: (33) 5 56 84 69 70. E-mail: [email protected]. (1) Maget-Dana, R. Biochim. Biophys. Acta 1999, 1462, 109. (2) Kawai, T.; Umemura, J.; Takenaka, T. Chem. Phys. Lett. 1989, 162, 243. (3) Castaings, N.; Blaudez, D.; Desbat, B.; Turlet, J. M. Thin Solid Films 1996, 284-285, 631.

Figure 1. Schematic view of a free-standing black film in a drilled porous glass plate.

Under these conditions, lipid black films could be a possible alternative. These free-standing films are made of two phospholipid monolayers separated by an interstitial water core of thickness less than 100 Å at the equilibrium. The polar headgroups of the lipid molecules are in contact with the aqueous phase and their tails face the ambient atmosphere (Figure 1). They form very easily from a drop of lipid vesicle solution in a hole drilled in a porous plate and are stable for hours and even days. Under high magnification microscope objectives (×50 or ×100), black films stay in focus, which is not the case for Langmuir monolayers that suffer from the evaporation of the subphase. For Raman spectroscopy, the main advantage of these black films is certainly that the intensity of the water bands is of the same order of magnitude (or even less) than those of the lipid monolayers, because of the nanometric thickness of the water core. At last, to study the effect induced by biologically active molecules, these molecules can be added to the vesicle solution before the formation of the black film. To date lipid black films have been rarely used to study protein-lipid interaction4,5 and no Raman study has been reported. As a preliminary step, we have previously studied black films of pure dimyristoylphosphatidylcholine (4) Lalchev, Z. I.; Todorov, R. K.; Christova, Y. T.; Wilde, P. J.; Mackie, A. R.; Clark, D. C. Biophys. J. 1996, 71, 2591. (5) Petkoval, V.; Nedyalkov, M.; Benattar, J. J. Colloids Surf. A 2001, 190, 9.

10.1021/la011247s CCC: $22.00 © 2002 American Chemical Society Published on Web 12/18/2001

Free-Standing Black Films

(DMPC)6 and followed the gel to LR liquid crystalline phase transition occurring at Tm ) 24 °C. The main result of this study is that the lateral interactions of the chains and the conformational order in the chains significantly decrease in the fluid LR liquid crystalline phase. In both phases, the thickness of the water core between the two monolayers was estimated to 22 ( 4 Å. In the present work, we try to validate these films for the study of protein-lipid interaction by Raman spectroscopy. We choose melittin, as the “peptide model” because of the abundant literature which exists on melittin-phospholipid interaction.1,7-20 First, we compare the quality of Raman spectra obtained from Langmuir monolayers and black films of DMPC. Then, we follow the effects induced on the phospholipid organization and on the water core thickness by the addition of increasing amounts of melittin in black films. Materials and Methods Materials. Dimyristoylphosphatidylcholine (DMPC) was purchased from Sigma. It was solubilized in ultrapure Millipore water (pH ) 5.5, resistivity > 18 MΩ‚cm) at a concentration of 0.5 mg/mL. Melittin is a hemolytic peptide of 26 amino acid residues which adopts in aqueous solution at high concentration or in membranes a structure of amphiphilic bent R-helix.21,22 Melittin was purchased from Serva (Serva Electrophoresis, Heidelberg, Germany). It was 99% pure and used without further purification. Methods. DMPC Langmuir Films. Langmuir films of DMPC were prepared and studied using a fully computerized 601M Nima trough. Following the usual method, DMPC was dissolved in chloroform at a concentration of 7 × 10-4 M and spread onto an ultrapure water subphase. The films were then compressed to a surface pressure of 30 mN/m at a constant speed barrier of 0.4 cm/min. Surface pressure was measured with a plate of filter paper held by a Wilhelmy balance (Nima) and kept constant at 30 mN/m during spectral acquisition. DMPC and Melittin)DMPC Black Films. Phospholipid black films were prepared following the standard procedure.23-25 Briefly, the DMPC aqueous solution was vortexed during 10 min, and then heated to 40 °C, above the gel to liquid crystalline LR phase transition temperature (Tm ) 24 °C). Melittin dissolved in water at a concentration of 10-4s10-3 M was added to the DMPC suspension at different melittin/DMPC molar ratios, Rm. This solution was next sonicated at room temperature during 20 (6) Lhert, F.; Capelle, F.; Blaudez, D.; Heywang, C.; Turlet, J.-M. J. Phys. Chem. B 2000, 104, 11704. (7) Stanislawski, B.; Ru¨terjans, H. Eur. Biophys. J. 1987, 15, 1. (8) Schwarz, G.; Beschiaschvili, G. Biochim. Biophys. Acta 1989, 979, 82. (9) Ladokhin, A. S.; Wimley, W. C.; White, S. H. Biophys. J. 1995, 69, 1964. (10) Rex, S.; Schwarz, G. Biochemistry 1998, 37, 2336. (11) Fattal, E.; Nir, S.; Parente, R. A.; Szoka, F. C. Biochemistry 1994, 33, 3, 6721. (12) Pott, T.; Paternostre, M.; Dufourc, E. J. Eur. Biophys. J. 1998, 27, 237. (13) Wackerbauer, G.; Weiss, I.; Schwarz, G. Biophys. J. 1996, 71, 1422. (14) Ohki, S.; Marcus, E.; Sukumaran, D. K.; Arnold, K. Biochim. Biophys. Acta 1994, 1194, 223. (15) Cornut, I.; Desbat, B.; Turlet, J.-M.; Dufourcq, J. Biophys. J. 1996, 70, 305. (16) Flach, C. R.; Prendergast, F. G.; Mendelsohn, R. Biophys. J. 1996, 70, 539. (17) Fidelio, G.; Maggio, B.; Cumar. F. A. Biochim. Biophys. Acta 1986, 862, 49. (18) Frey, S.; Tamm, L. K. Biophys. J. 1991, 60, 922. (19) Schoch, P.; Sargent, D. F. Biochim. Biophys. Acta 1980, 602, 234. (20) Tosteson, M. T.; Tosteson, D. C.. Biophys. J. 1981, 36, 109. (21) Akyu¨z, S.; Davies, J. E. D. Vibr. Spectrosc. 1993, 4, 199. (22) Lavialle, F.; Adams, R. G.; Levin, I. W. Biochem. 1982, 21, 2305. (23) Mysels, K. J.; Shinoda, K.; Frankel, S. Soap films, studies of their thinning; Pergamon: New York, 1959. (24) Bergeron, V.; Radke, C. J. Langmuir 1992, 8, 3020. (25) Capelle, F.; Lhert, F.; Blaudez, D.; Kellay, H.; Turlet, J.-M. Colloids Surf. A 2000, 171, 199.

Langmuir, Vol. 18, No. 2, 2002 513 min, and unilamellar vesicles were obtained with a typical radius of 75 nm as measured by dynamic light scattering. To form the film, drops of the mixed melittin-DMPC solution were deposited at the level of a hole of about 1 mm in diameter drilled in a porous glass disk.25 Raman Spectroscopy. As previously described,25 Raman spectra of black films were recorded with a confocal micro-Raman set up based on a T64000 spectrometer (Jobin Yvon) equipped with an Olympus BH2 microscope. The excitation was realized with the line at λ ) 514.5 nm of a Coherent Innova 305 Ar+ laser. The beam was focused on the sample with a long working distance 100× microscope objective (Olympus ULWD-MS-Plan). The scattered light was retro-collected by the same objective and passed through a 200 µm pinhole aperture located in the conjugated plane. Under these confocal optical conditions, the axial resolution was equal to 4 ( 0.4 µm.25 After rejection of the Rayleigh component through a holographic notch filter (Kaiser Optical Systems), the Raman scattered light was spectrally analyzed on the final spectrograph stage of the T64000 spectrometer, equipped with a 600 g/mm grating and a EEV 05-10 liquid nitrogen cooled CCD detector. With a 100 µm entrance slit, the spectral resolution was 8 cm-1. The cell containing the porous disk was directly sealed by the microscope objective. This enabled to keep the film at a constant humidity rate. The temperature was kept constant at 29 ( 1 °C (i.e., above the gel to LR phase transition of DMPC) in order to have fluid chains. One had to wait for 24-48 h to let the film become homogeneous and stable in thickness. For monolayer studies, the Langmuir trough was equipped with a cover in which holes were drilled to allow the passage of the objective and of the filter paper of the balance. This cover had to be open on one side for barrier movement, and thus, a constant humidity was not achieved. This resulted in evaporation which induced a slight defocusing of the laser beam on the monolayer (typically a few micrometers after the 10 min recording). Monolayer and black film spectra have been recorded using the same acquisition conditions. Because of the weakness of the Raman signal, it was necessary to co-add about 12 spectra, which corresponds to a total acquisition time of 2 h.

Results (a) Raman Spectra of Langmuir Monolayers. In Figure 2a is presented the Raman spectrum of a DMPC Langmuir monolayer at a surface pressure of 30 mN/m. In this spectrum are predominant the two contributions of the liquid water subphase: the broad and intense νOH band centered at 3400 cm-1 (spectral window has been shifted to avoid saturation of the CCD response) and the δ(OH2) mode located at 1650 cm-1. One also can make a remark on the contributions of atmospheric nitrogen and oxygen at 2300 and 1555 cm-1, respectively. The ν(CH2) bands of the DMPC monolayer are hardly distinguishable as a very small bump on the wing of the strong νOH band. After subtraction of the background due to the pure water subphase, the Raman spectrum of the DMPC monolayer is revealed (Figure 2b). However, despite the very long acquisition time (about 2 h), this spectrum is still noisy. If the intensity of the ν(CH2) bands can be reasonably estimated, conversely the modes of the low-frequency region cannot be neatly extracted from the noise. This restricts quantitative analysis and makes Raman study of protein-lipid interaction in monolayers very difficult. (b) Raman Spectra of Black Films. Figure 3 shows the Raman spectrum of a melittin-DMPC black film at a melittin/DMPC molar ratio Rm (ratio in the solution before the formation of the film) equal to 1/80. In both spectral regions this spectrum presents a very good signalto-noise (S/N) ratio which allows unambiguous identification of the vibrational modes of DMPC. These modes have

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Figure 2. Raman spectrum of a DMPC monolayer at the air/water interface before (a) and after (b) subtraction of the subphase signal.

Figure 3. Raman spectrum in the 4000-2600 (a) and 1850-650 cm-1 (b) spectral ranges of a melittin-DMPC black film at a peptide/lipid molar ratio Rm ) 1/80.

been previously described in details.26-28 Briefly, in the 3100-2700 cm-1 range (Figure 3a), it is possible to observe the C-H stretching vibrations of the DMPC alkyl chains. The methylene groups of the chains give rise to two intense bands at 2853 and 2885 cm-1 corresponding to the symmetric νs(CH2) and antisymmetric νa(CH2) stretching modes, respectively. The broad shoulder at 2925 cm-1 is due to the overtone of the CH2 scissoring δ(CH2) enhanced by Fermi resonance with the νs(CH2) vibration, and to the νs(CH3) vibration. The band at 2965 cm-1 is assigned to the antisymmetric stretching νa(CH3) of the methyl groups located at the end of the chains, whereas the band at 3042 cm-1 is assigned to the stretching mode of the choline methyl of polar headgroups. In the 1850-650 cm-1 spectral range (Figure 3b), the bands located at 1441 and 1300 cm-1 are assigned to the methylene scissoring δ(CH2) and twisting t(CH2) modes, respectively. The two bands located at 1124 and 1063 cm-1 (26) Gaber, B. P.; Peticolas, W. L. Biochim. Biophys. Acta 1977, 465, 260. (27) Snyder, R. G.; Schachtschneider, J. H. Spectrochim. Acta 1963, 19, 85. (28) Levin, I. W. In Advances in Infrared and Raman Spectroscopy; Clark, R. J. H., Hester, R. E., Eds.; Wiley Heyden: New York, 1984; Volume 11, Chapter 1, pp 1-48.

are assigned to the in-phase and out-of-phase skeletal stretching vibrations of the C-C bonds, respectively.27,28 The former only arises from all-trans bonds27 while the latter is moderately sensitive to the presence of gauche conformational defects.28 The band at 1088 cm-1 results from the superposition of the C-C stretching vibration mode with some other vibrational modes; it has been shown to be sensitive to the presence of different kinds of gauche conformers in the alkyl chains.28-30 The band at 1736 cm-1 corresponds to the stretching vibration of the CdO ester groups, and the band at 875 cm-1 is assigned to the antisymmetric stretching of the C-N bonds νa(CN).28 The bands at 770 and 716 cm-1 are associated with trans and gauche conformations of the -O-C-C-N+ group, respectively. The spectral signature of the interstitial aqueous core appears in the 3800-3100 cm-1 range (Figure 3a) as a broad and multicomponent band centered at 3400 cm-1. The assignment of this band is still controversial. Usually it is divided in three main components. Some authors31,32 (29) Snyder, R. G. J. Chem. Phys. 1967, 47, 1316. (30) Zerbi, G.; Magni, R.; Gussoni, M.; Holland-Moritz, K.; Bigotto, A.; Dirlikov, S. J. Chem. Phys. 1981, 75, 3175. (31) Sokolowska, A.; Kecki, Z. J. Raman Spectrosc. 1986, 17, 29.

Free-Standing Black Films

Figure 4. Raman spectra in the 3800-2700 cm-1 spectral range of melittin-DMPC black films at different molar ratios (Rm ) 0, 1/1000, 1/500, 1/250, 1/80, 1/55). Arrows indicate how the bands are varying when Rm is increased. (Spectra at Rm ) 0 and Rm ) 1/ 1000 are identical.)

assigned these components to the symmetric OH stretching in Fermi resonance interaction with the overtone of the δ(OH2) bending mode (low-frequency component), the antisymmetric OH stretching (medium-frequency component), and the OH stretching of free OH bonds (highfrequency component). Conversely, an assignment in terms of “network water”, “intermediate water”, and “multimer water” molecules has been proposed33 for the low-, medium-, and high-frequency components, respectively. However, it is well-established that the intensity profile of this band varies with the kinds and degrees of organization that water molecules exhibit.34 Therefore, we used the position of the barycenter (intensity weighted center) of the whole band to monitor possible modifications of water structure in the film with respect to the bulk isotropic water structure. Figures 4 and 5 show the evolution of the Raman spectra of melittin-DMPC black films when the molar ratio Rm is increased from 0 to 1/55. Whatever the concentration is, no specific band of melittin is detected since the number of melittin molecules in the film is low and its modes are weakly Raman active. When Rm is lower than 1/500 almost no modification on the DMPC Raman spectra is observed, as shown for instance in the spectrum observed at Rm ) 1/1000. Conversely, gradual changes are observed in the 3100-2700 cm-1 spectral range for melittin/DMPC ratios higher than 1/ 500. In Figure 4, all spectra have been normalized with respect to the νs(CH2) band at 2850 cm-1 since the variation in intensity of this band is small under different experimental conditions. As indicated by the black arrows, when Rm increases, the intensity of the νa(CH2) band at 2885 cm-1 grows up while the intensities of the bands at 2925 and 2965 cm-1 decrease. Modifications are also observed in the 1850-650 cm-1 spectral range (Figure 5). In this region, spectra are normalized with respect to the band at 720 cm-1 (the intensity of this band does not change with temperature). (32) Rull, F.; de Saja, J. A. J. Raman Spectrosc. 1986, 17, 167. (33) Brubach, J. B.; Mermet, A.; Filabozzi, A.; Gerschel, A.; Lairez, D.; Krafft, M. P.; Roy, P. J. Phys. Chem. B 2001, 105, 430 and references therein. (34) Lafleur, M.; Pigeon, M.; Pe´zolet, M.; Caille´, J.-P. J. Phys. Chem. 1989, 93, 1522 and references therein.

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Figure 5. Raman spectra in the 1800-650 cm-1 spectral range of melittin-DMPC black films at different molar ratios (Rm ) 0, 1/500, 1/80). Arrows indicate how the bands are varying when Rm is increased.

When Rm increases, bands at 1124 (νs(C-C)trans) and 1441 cm-1 (δ(CH2)) increase and the intensity of the band at 1300 cm-1 (t(CH2)) decreases while the intensity of the band at 1088 cm-1 sensitive to the C-C vibrations of gauche conformers remains almost constant. Regarding the amount of water in the film, one can see in Figure 4 that the broad band of water becomes more intense when Rm increases, showing that the thickness of the water core increases. For ratios Rm higher than 1/55, we have to mention that a huge swelling of the film occurs. The intensity of the water band drastically increases with a corresponding thickness of the water core situated in the micrometer range. Discussion Comparison of Figures 2 and 3 clearly shows that the black film configuration gives much better Raman spectra than Langmuir monolayers. Quantitatively, all band intensities are about 5 times smaller in the monolayer spectrum instead of the expected factor of 2 (going from a bilayer to a monolayer). This is due to the continuous defocusing of the laser beam on the monolayer during the 10 min acquisition time. Moreover, in the monolayer spectrum, the subtraction of the strong and broad water subphase background contributes to a decrease of the S/N ratio. An important consequence of this low S/N ratio is that bands located below 1200 cm-1 cannot be neatly extracted from the noise. Therefore, a full spectral analysis of the changes induced by melittin cannot be reasonably undertaken with monolayer samples and the following discussion will be restricted to black film samples. Raman spectra give information on the lateral order in phospholipid systems. In particular the peak height ratio F ) Iva(CH2)/Ivs(CH2) is representative of chain packing and chain mobility.35 As an indication, F is equal to 0.7 when the chains are completely fluid (liquid state) and equal to 2.2 in the case of a perfect crystalline state.26 From Raman spectra one can also deduce information on the conformational order of the alkyl chains. Pink et al.36 proposed to probe the fluidity of the hydrocarbon chains using the integrated intensities of the νs(C-C)trans (35) Snyder, R. G.; Scherer, J. R.; Gaber, B. P. Biochim. Biophys. Acta 1980, 601, 47. (36) Pink, D. A.; Green, T. J.; Chapman, D. Biochemistry 1980, 19, 349.

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Table 1. Thickness of the Water Layer and Values of G and Df Parameters Calculated from Raman Spectra of Black Films of DMPC and Melittin/DMPC at Different Melittin/DMPC Molar Ratios (Rm) melittin/DMPC molar ratio, Rm

water core thickness (Å)

F

Df

0(DMPC in gel phase) 0(DMPC in LR phase) 1/1000 1/500 1/250 1/80 1/55

22 ( 5 22 ( 5 25 ( 5 47 ( 5 110 ( 12 262 ( 27 397 ( 41

1.36 1.06 1.07 1.22 1.27 1.31 1.36

0.41 0.33 0.36 0.40 0.49 0.66 0.71

and ν(CN) bands located at 1124 and 716 cm-1, respectively. The degree of fluidity of a sample is defined as Df ) (I1124/I716)sample/(I1124/I716)solid where (I1124/I716)solid is the intensity ratio measured on a bulk spectrum recorded at low temperature (T ) -40 °C). In fact, Pink et al.36 have shown that it is necessary to lower the temperature down to -100 °C to get a perfect all-trans organization in DMPC chains. However, the same study and a work of Bertolluza et al.37 have shown that the number of gauche defects is still very low until -40 °C, which is a temperature much more convenient for Raman micro-spectroscopy. Conversely, in the gel phase at 19 °C, DMPC chains present a much higher number of defects36 which makes this gel phase not suitable as a reference for perfect conformational order. As can be seen in Figures 4 and 5, no modification of the DMPC black film Raman spectrum is observed for the lowest molar ratio Rm ) 1/1000. As described in the Experimental Section, Rm is the melittin/DMPC molar ratio in the solution before the formation of the film. Therefore, it may not reflect exactly the actual concentration in the film but accurate measurement of this concentration would be difficult. Schwarz and Beschiaschvili8 studied the binding of melittin to DOPC unilamellar vesicles by circular dichroism. They described this binding as a partitioning of the peptide between aqueous and phospholipid phases, melittin dissolving in the lipid phase because of favorable solvation effects. Extrapolating their results to our experimental conditions, the percentage of bound melittin molecules would increase from 80% to 100% when Rm decreases from 1/55 to 1/1000. Thus, melittin is supposed to be mainly associated with lipid vesicles under our conditions. As black films directly form from vesicles one can tentatively assume that Rm is also indicative of the final melittin/DMPC molar ratio in the film. At ratios as low as 1/1000, the number of melittin molecules in the film is likely too low to induce an effect on the phospholipid conformation and on the structure of the film. Correlatively the thickness of the water core is the same than in the pure DMPC black films (about 25 Å) within the experimental uncertainties. As reported in Table 1, the F and Df parameters at Rm ) 1/1000 are very close to those found in pure DMPC black films and confirm that melittin at this very low concentration has no effect on the lateral and conformational organization of the film. Significant modifications occur in the spectra for Rm ) 1/ 500 and become more pronounced as the concentration of melittin is increased up to Rm ) 1/55. This observation shows that increasing amounts of melittin are present in the films when Rm increases. In the LR phase at 29 °C, the F parameter (Table 1) which is characteristic of the lateral order of the DMPC chains, grows up from 1.06 for Rm )

0 to 1.36 for Rm ) 1/55; so, the lateral order of DMPC chains is higher in the presence of melittin. It is worth noting that this last value of F is equal to the one found in pure DMPC black films in the gel phase at 19 °C. Thus, the presence of increasing concentrations of melittin in the film has the same effect on the lateral order of the phospholipid than a continuous decrease in temperature. The same behavior is observed for the Df parameter, characteristic of the conformational order of the aliphatic chains (see Table 1). Its value increases from 0.33 for Rm ) 0 to 0.71 for Rm ) 1/55 which shows that melittin strongly reduces the number of gauche defects in DMPC black films in the LR phase. As Df is equal to 0.41 for pure DMPC in the gel phase, we can conclude that melittin has a much stronger effect on the conformational order of phospholipid than a simple temperature lowering. This ordering effect of DMPC induced by melittin agrees with some observations done on Langmuir monolayers. Indeed, it has been shown by IR spectroscopy that melittin induces a gauche to trans isomerization in DPPC monolayers and decreases the motional rates of alkyl chains.16 Surface pressure measurements also showed that the interaction of melittin with DMPC monolayers increases the stability of both molecules.17 On the other hand, it has been reported that melittin increases the conformational disorder of phospholipids in a bilayer configuration such as small unilamellar vesicles21 or bilayer suspensions.38 As far as these discrepancies are not due to different experimental conditions (temperature, concentration, pH, ionic strength, ...) they could indicate that black films are closer from the monolayer than from a bilayer configuration. Also, melittin induces effects on the water core thickness of the black film (Figure 4). The intensity of the water band at 3400 cm-1 enables to estimate the thickness of the aqueous core between the two monolayers by comparing this intensity with the one observed with a thick sample of liquid water.25 In Figure 4, one can observe that the intensity of the broad water band increases with the peptide to lipid molar ratio Rm from 1/500. The corresponding thickness values of the water core are reported in Table 1 and plotted vs Rm in Figure 6. This thickness increases almost linearly with the amount of melittin from 22 Å at Rm ) 0 to about 400 Å at Rm ) 1/55. In the frame of the

(37) Bertolluza, A.; Bonora, S.; Fini, G.; Morelli, M. A. Can. J. Chem. 1985, 63, 1390.

(38) Dasseux J.-L.; Faucon, J.-F.; Lafleur, M.; Pe´zolet, M.; Dufourcq, J. Biochim. Biophys. Acta 1984, 775, 37.

Figure 6. Water core thickness in melittin/DMPC black films plotted vs melittin/DMPC molar ratio Rm.

Free-Standing Black Films

Figure 7. Raman spectrum of a melittin-DMPC black film at a peptide/lipid molar ratio Rm ) 1/150 with (a) and without (b) electrolyte (500 mM NaCl).

DLVO (Derjaguin, Landau, Verwey, and Overbeek) theory, the equilibrium water core thickness of a black film is governed by the balance between attractive van der Waals interaction between the two monolayers and repulsive electrostatic double-layer forces.39,40 This repulsive force develops due to the entropic confinement of the counterions which neutralize the charged interface. As melittin is strongly positively charged (six positive charges under our experimental conditions), the number of Cl- counterions rapidly increases with melittin concentration, and the equilibrium aqueous core thickness shifts toward higher values. It is well-known that the addition of electrolyte in ionic surfactant black films partially screens the electrostatic double-layer forces and results in a thinning of the water core.23,24 To verify that a mixed melittin-DMPC black film behaves similarly, we have recorded the Raman spectrum of a mixed film made from a solution (Rm ) 1/150) containing 500 mM of NaCl (Figure 7). Clearly, with the addition of salt, the amount of water in the black film drastically decreases. Quantitatively the aqueous core thickness drops from 104 to 28 Å. This last value is close to the one obtained (about 20 Å) with the sodium dodecyl sulfate (SDS) cationic surfactant in the presence of electrolyte.24,25 Also, within the experimental uncertainties, the water core in the mixed film in the presence of salt is still a little bit thicker than the one in a pure DMPC film (22 Å). This shows that the electrostatic double-layer repulsion between two monolayers of zwitterionic molecules is weak. The spectral shape of the water band is also varying according to the melittin/DMPC ratio. In Figure 8 are reported the Raman spectra of the water core of black films with two different Rm values and the spectrum of pure liquid water. All spectra have been normalized in intensity with respect to the high-frequency component. Going from bulk water to the pure phospholipid black film where the water core thickness is minimum (22 Å), one observes a decrease of the relative intensity of the low-frequency component at 3240 cm-1. Correlatively the intensity weighted barycenter of the whole band shifts from 3367 cm-1 for pure water to 3389 cm-1 for the pure (39) Israelachvili, J. N. Intermolecular and Surface Forces with Applications to Colloid and Biological Systems; Academic: Orlando, FL, 1985. (40) Bergeron, V. J. Phys.: Condens. Matter 1999, 11, R215.

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Figure 8. Shape of the multicomponent water band in the case of bulk water (thick solid line), pure DMPC black film (thin solid line) and melittin-DMPC black film with Rm ) 1/250 (dotted line).

DMPC black film aqueous core. For Rm )1/250 and a water core thickness of 110 Å it sets to an intermediate value of 3375 cm-1. Such a decrease has been observed by Lafleur et al.34 in hydrated DPPC multilamellar bilayers and has been attributed to a confinement of water molecules between the two polar head planes of the lipids (in this case the water thickness was equal to 17 Å). Thus, one can state that the shift of the barycenter of the water band represents a good probe of the confinement of water molecules in black films. When the ratio Rm is higher than 1/55, a huge swelling of the film is observed leading to a water core thickness of several micrometers. At such high ratios the formation of melittin-phospholipid co-micelles is supposed to occur with zwitterionic phospholipids.41 Thus, co-micelles could form in the aqueous core of the black film. As these comicelles are strongly charged because of the presence of melittin, an additional electrostatic repulsion, significantly higher than the repulsive electrostatic double-layer forces would occur and the equilibrium water core thickness would increase drastically. Conclusion In this report, free-standing black films made of two monolayers of phospholipid separated by a nanometric interstitial water core have been considered to study lipidprotein interaction by Raman spectroscopy. Spectra obtained from black films present a much higher S/N ratio than Langmuir monolayer spectra obtained under the same conditions. This difference in quality is mainly due to the absence of screening effect of the aqueous subphase signal together with twice a population of probed molecules. It allows accurate quantitative analysis in terms of molecular organization. We took benefit of this advantage to study the effects induced by melittin on DMPC black films. These effects have been observed even at a melittin/phospholipid molar ratio in the solution as low as 1/500. This concentration threshold and, more generally, the low melittin/DMPC ratios used in this study are significantly lower than the values usually met in the literature, and underline the sensitivity of Raman spectroscopy to detect subtle changes in the lipid conformation. (41) Dufourc, E. J.; Faucon, J.-F.; Fourche, G.; Dufourcq, J.; GulikKrzywicki, T.; Le Maire, M. FEBS Lett. 1986, 201, 205.

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In particular, we observed that the presence of melittin in the film induces an increase of the lateral and conformational order of the DMPC chains. Since these conclusions go the same way as those previously reported for Langmuir monolayers, some similarity between these two complementary configurations can be inferred. Comparing these two ultrathin systems, Langmuir monolayers allow easy variation and monitoring of molecular packing whereas black films have the great advantage for optical studies of not generating any background signal. More work has to be done to accurately determine protein

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concentration in these films and to observe directly the Raman signature of proteins. Of course black films, as Langmuir monolayers, are not biological membranes, but structural and dynamical studies of protein interactions on these ultrathin lipid systems can certainly help to understand basic phenomena occurring in the “real world”. Acknowledgment. We thank the “Aquitaine Region” research council for financial support and Pr. Alois Wu¨rger for useful discussions on DLVO theory. LA011247S