Interaction of Primary Amphipathic Cell-Penetrating Peptides with

The mesoscopic organization adopted by two primary amphipathic peptides, P and P(α), in Langmuir−Blodgett (LB) films made of either the pure peptid...
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Langmuir 2004, 20, 9255-9261

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Interaction of Primary Amphipathic Cell-Penetrating Peptides with Phospholipid-Supported Monolayers Thomas Ple´nat,† Se´bastien Deshayes,‡ Sylvie Boichot,† Pierre Emmanuel Milhiet,† Richard B. Cole,§ Fre´de´ric Heitz,‡ and Christian Le Grimellec*,† Nanostructures et Complexes Membranaires, CBS, CNRS UMR5048-INSERM U554, 29 rue de Navacelles, 34090 Montpellier Cedex, France, CRBM, CNRS-FRE 2593, IFR 122, 34293 Montpellier Cedex, France, and Department of Chemistry, University of New Orleans, New Orleans, Louisiana 70148 Received June 4, 2004. In Final Form: July 27, 2004 The mesoscopic organization adopted by two primary amphipathic peptides, P(β) and P(R), in LangmuirBlodgett (LB) films made of either the pure peptide or peptide-phospholipid mixtures was examined by atomic force microscopy. P(β), a potent cell-penetrating peptide (CPP), and P(R) mainly differ by their conformational states, predominantly a β-sheet for P(β) and an R-helix for P(R), as determined by Fourier transform infrared spectroscopy. LB films of pure peptide, transferred significantly below their collapse pressure, were characterized by the presence of supramolecular structures, globular aggregates for P(β) and filaments for P(R), inserted into the monomolecular film. In mixed peptide-phospholipid films, similar structures could be observed, as a function of the phospholipid headgroup and acyl chain saturation. They often coexisted with a liquid-expanded phase composed of miscible peptide-lipid. These data strongly suggest that primary amphipathic CPP and antimicrobial peptides may share, to some extent, common mechanisms of interaction with membranes.

Introduction Transport across the cell plasma membrane remains a serious obstacle in the development of peptide-, protein-, and nucleic acid-based drugs and their therapeutic use.1-3 In recent years several carrier peptides, also called cellpenetrating peptides (CPPs), have been demonstrated to translocate cargo across the plasma membrane of eukaryotic cells.4-6 The mechanisms involved in the peptide/cargo translocation across the cell plasma membrane, however, remain a matter of debate. Thus, whereas a receptor-mediated process can be excluded, it is not clear to what extent some of the CPPs require energy to enter the cell.7-9 As compared to the antibiotic peptide family, which might form “carpetlike” structures that destabilize the bilayer organization or induce the formation of “porelike” structures,10-13 the interactions of CPP with membrane lipids are still poorly characterized. Most of * To whom correspondence should be addressed. Phone: (33) 467 41 79 07. Fax: (33) 467 41 79 13. E-mail: [email protected]. † CBS CNRS UMR5048-INSERM U554. ‡ CNRS-FRE 2593. § University of New Orleans. (1) Gewirtz, A. M.; Sokol, D. L.; Ratajczak, M. Z. Blood 1998, 92, 712. (2) Juliano, R. L.; Alahari, S.; Yoo, H.; Kole, R.; Cho, M. Pharm. Res. 1999, 16, 494. (3) Lindgren, M.; Ha¨llbrink, M.; Prochiantz, A.; Langel, U. TIPS 2000, 21, 99. (4) Derossi, D.; Chassaing, G.; Prochiantz, A. Trends Cell Biol. 1998, 8, 84. (5) Ford, K. G.; Souberbielle, B. E.; Darling, D.; Farzaneh, F. Gene Ther. 2001, 8, 1-4. (6) Jarver, P.; Langel, U. Drug Discovery Today 2004, 9, 395. (7) Drin, G.; Cottin, S.; Blanc, E.; Rees, A. R.; Temsamani, J. J. Biol. Chem. 2003, 278, 31192. (8) Richard, J. P.; Melikov K.; Vives, E.; Ramos, C.; Verbeure B.; Gait, M. J.; Chernomordik, L. V.; Lebleu, B. J. Biol. Chem. 2003, 278, 585. (9) Tre´hin, R.; Krauss, U.; Muff., R.; Meinecke, M.; Beck-Sickinger, A. G.; Merkle, H. P. Pharm. Res. 2004, 21, 33. (10) Epand, R. M.; Shai, Y.; Segrest, J. P.; Anantharamaiah, G. M. Biopolymers 1995, 37, 319. (11) Matsuzaki, K. Biochim. Biophys. Acta 1998, 1376, 391.

the lipid-CPP interaction studies concern the Antennapedia-derived CPP penetratin, a 16-residue fragment that has proven to be an excellent transport vector.3,4 Recent results suggest that this CPP can translocate through lipid bilayers by a potential- and lipid compositiondependent pathway which could involve a local electroporation mechanism.14-17 Members of another family of CPPs, based on the design of synthetic primary amphipathic peptides, were shown to act as efficient drug carriers, resulting in a rapid internalization process.18,19 The structure of one of these CPPs was based on the association of a hydrophobic 20residue signal peptide20 with a sequence issued from a positively charged nuclear localization motif.21,22 Analysis of peptide-phospholipid mixed monolayers showed that this peptide interacted with both zwitterionic and negatively charged phospholipids and suggested an R to β conformational transition, associated with the formation of peptide-containing filaments, upon an increase of the peptide molar fraction.23,24 Replacing the signal peptide sequence by a hydrophobic 17-residue sequence derived (12) Yang, L.; Harroun, T. A.; Weiss, T. M.; Ding. L.; Huang, H. W. Biophys. J. 2001, 81, 1475. (13) Shai, Y. Biopolymers 2002, 66, 236. (14) Drin, G.; De´me´ne´, H.; Temsamani, J.; Brasseur, R. Biochemistry 2001, 40, 1824. (15) Binder, H., Lindblom, G. Biophys. J. 2003, 85, 982. (16) Terrone, D.; leung Wai Sang, L.; Roudaia, L.; Silvius, J. R. Biochemistry 2003, 42, 13787. (17) Ziegler, A.; Li Blatter, X.; Seelig, A.; Seelig, J. Biochemistry 2003, 42, 9185. (18) Vidal, P.; Chaloin, L.; Me´ry, J.; Lamb, N.; Lautredou, N.; Bennes, R.; Heitz, F. J. Pept. Sci. 1996, 2, 125. (19) Chaloin, L.; Vidal, P.; Lory, P.; Me´ry, J.; Lautredou, N.; Divita, G.; Heitz, F. Biochem. Biophys. Res. Commun. 1998, 243, 601. (20) Briggs, M. S.; Gierasch, L. M. Adv. Protein Chem. 1986, 38, 109. (21) Kalderon, D.; Richardson, W. D.; Markham, A. F.; Smith, A. E. Nature 1984, 311, 33. (22) Goldfarb, D. S.; Garie´py, j.; Schoolnik, G.; Kornberg, R. D. Nature 1986, 322, 641. (23) Van Mau, N.; Vie´,V.; Chaloin, L.; Lesniewska, E.; Heitz, F.; Le Grimellec, C. J. Membr. Biol. 1999, 167, 241.

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from the protein gp41 from human immunodeficiency virus (HIV) gave a CPP (CPP-HIV) also having a strong ability to deliver nucleic acids into cells in culture.25-27 This peptide is nonstructured in water, but the presence of phospholipids induces formation of a sheet structure.28,29 To better characterize how these primary amphipathic CPPs interact with phospholipids, we synthesized two peptides closely related by their sequence but capable to adopt different conformational states. The first one, called hereafter peptide P(β), has conformational properties similar to those of the parent peptide CPP-HIV, differing by a single W7 to F7 substitution, leading to sequence A. Peptide P(R) (sequence B)

was designed using the AGADIR algorithm as the peptide having a sequence as close as possible to that of P(β) but with a larger helical content.30 For this peptide, the predicted helical content in the 1-16 region is about 13%, which has to be compared with 1% for P(β). In recent years, atomic force microscopy (AFM) was shown to provide unique information on peptide-peptide and lipidpeptide interactions in both monolayers and supported bilayers.23,24,31-34 Accordingly, in the present experiments we use AFM to characterize the behavior of LangmuirBlodgett films made of P(β) and P(R) alone, and in the presence of zwitterionic and negatively charged phospholipids.

Figure 1. Surface pressure vs area per molecule isotherms for P(β) (a) and P(R) (b) at the air-water interface. Isotherms were obtained at room temperature.

Materials and Methods Peptides P(β) and P(R) were synthesized by solid-phase peptide synthesis using AEDI-Expansin resin on a Pioneer peptide synthesizer (Applied Biosystems, Foster City, CA) according to the Fmoc/tBoc method, as already described.18 Peptides were acetylated at their N-terminus, and both have a cysteamide group at their C-terminus. Dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylcholine (DPPC), and dipalmitoylphosphatidylglycerol (DPPG) were purchased from Avanti Polar Lipids (Alabaster, AL) and dissolved in a chloroform/methanol solution (3:1, v/v) at a concentration of 1 mM. The peptides were dissolved in DMSO/chloroform/ methanol mixtures (0.03:3:1, v/v/v). Monolayers were spread, at room temperature, on high-purity water (triply distilled water on MnO4K). Lipid-peptide mixtures were prepared at the desired compositions and spread on the water, and then the solvent was allowed to evaporate for at least 10 min before the compression was started, at a rate of 0.015 (nm2/molecule)/min. Interfacial pressures were measured with a Wilhelmy balance using a platinum plate. All LangmuirBlodgett (LB) transfers were achieved at a constant surface (24) Vie´, V.; Van Mau, N.; Chaloin, L.; Lesniewska, E.; Le Grimellec, C.; Heitz, F. Biophys. J. 2000, 78, 846. (25) Morris, M. C.; Vidal, P.; Chaloin, L.; Heitz, F.; Divita, G. Nucleic Acids Res. 1997, 25, 2730. (26) Morris, M. C.; Chaloin, L.; Mery, J.; Heitz, F.; Divita, G. Nucleic Acids Res. 1999, 27, 3510. (27) Simeoni, F.; Morris, M. C.; Heitz, F.; Divita, G. Nucleic Acids Res. 2003, 31, 2717. (28) Vidal, P.; Chaloin, L.; Heitz, A.; Van Mau, N.; Me´ry, J.; Divita, G.; Heitz; F. J. Membr. Biol. 1998, 162, 259. (29) Pereira, F. B.; Goni, F. M.; Muga, A.; Nieva, J. L. Biophys. J. 1997, 73, 1977. (30) Munoz, V.; Serrano, L. Biopolymers 1997, 41, 495. (31) Mou, J.; Czajkowsky, D. M.; Shao, Z. Biochemistry 1996, 35, 3222. (32) Rinia, H. A.; Kik, R. A.; Demel, R. A.; Snel, M. M. E.; Killian, J. A.; van der Eerden, J. P. J. M.; de Kruijff, B. Biochemistry 2000, 39, 5852. (33) Steinem, C.; Galla, H.-J.; Janshoff, A. PhysChemPhys 2000, 2, 4580. (34) Diociaiuti, M.; Bordi, F.; Motta, A.; Carosi, A.; Molinari, A.; Arancia, G.; Coluzza, C. Biophys. J. 2002, 82, 31998.

Figure 2. FTIR spectra in the amide I region of pure peptide (A) and peptide-phospholipid mixtures (B). Spectra a and b correspond to P(R) and P(β), respectively. In the presence of phospholipid, peptide:lipid ratios are 1:1 with DOPG for P(R) and DOPC for P(β). Similar spectra are obtained for DPPC and DPPG. pressure of 26 mN/m, after a 5 min relaxation period, by raising vertically (5 mm/min) freshly cleaved mica through the airwater interface.35 Fourier transform infrared (FTIR) spectra were obtained on a Bruker IFS 28 spectrometer equipped with a liquid nitrogen cooled MCT detector. Spectra (1000-2000 scans) were recorded at a spectral resolution of 4 cm-1 and were analyzed using the OPUS/IR2 program. Samples were obtained by deposition of solutions of lipid and peptide mixtures onto a fluorine plate where the solvents were allowed to evaporate under a nitrogen flux.23 AFM imaging of LB films was performed in contact mode using a Nanoscope IIIa atomic force microscope (Digital Instruments, (35) Vie´, V.; Van Mau, N.; Lesniewska, E.; Goudonnet, J. P.; Heitz, F.; Le Grimellec, C. Langmuir 1998, 14, 4574.

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Figure 3. AFM topographic images of P(β) (A, B) and P(R) (C, D) monolayers. Scan size: 5 µm (A), 1 µm (B), 3 µm (C) 1 µm (D). Scan rate: 1.5 Hz. Vertical (z) scale: 5 nm (A), 2.5 nm (B-D). (E) is a virtual section of a zoom of (D). Santa Barbara, CA) under ambient conditions using a 120 µm (J) scanner. Topographic images were acquired in constant mode using silicon nitride tips on integral cantilevers with a nominal spring constant of 0.12 N/m. Typically, the estimated imaging forces were below 0.5 nN.36 Images were obtained from at least two different samples prepared on different days with at least five macroscopically separated areas on each sample. Representative images are presented.

Results Imaging of Pure Peptide Monolayers. The compression isotherm of P(β), with no evidence for collapse up to a pressure of ∼40 mN/m, was comparable (Figure 1, trace a) to that reported for another β-sheet-forming primary amphipathic peptide.23,24 On the other hand, in terms of hydrophobicity, the limited modification of the P(R) hydrophobic sequence generated by the replacement of G6, G9, G12, T14, and A17 by A6, A9, L12, L14, and L17, (36) Giocondi, M.-C.; Vie´, V.; Lesniewska, E.; Milhiet, P. E.; ZinkeAllmang, M.; Le Grimellec C. Langmuir 2001, 17, 1653.

which promoted a helical conformation (see below), resulted in a steeper pressure vs molecular area curve, i.e., a more rigid monolayer corresponding to stronger peptide-peptide interactions, with a collapse pressure at ∼32 mN/m (Figure 1, trace b). This value is close to the estimate of a surface pressure equivalent in biological membranes.37,38 Accordingly, tranfers were performed at 26 mN/m to limit possible artifacts upon transfer onto the solid support,39,40 while remaining reasonably close to the physical conditions of biological membranes. The infrared spectra, which showed a major amide I band component centered around 1625-1630 nm cm-1, confirmed the (37) Philips, M. C.; Williams, R. M.; Chapman, D. Chem. Phys. Lipids 1969, 3, 234. (38) Demel, R. A.; Geurts von Kessel, W. S. M.; Zwaal, R. F. A.; Roelfson, B.; van Deenen, L. L. M. Biochim. Biophys. Acta 1975, 406, 97. (39) Seul, M.; Subramaniam, S.; McConnell, H. J. Phys. Chem. 1985, 89, 3592. (40) Lee, K. Y. C.; Lipp, M. M.; Takamoto, D. Y.; Ter-Ovanesyan, E.; Zasadzinski, J. A. Langmuir 1993, 9, 2567.

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Figure 4. Low-magnification imaging of mixed peptide-saturated phospholipid films at low peptide concentration (5 mol %): (A) P(β)-DPPC; (B) P(R)-DPPC; (C) P(β)-DPPG; (D) P(R)-DPPG. Scan size: 5 µm.

existence of a dominant conformation corresponding to a β-type for P(β) (Figure 2A, trace b). In accordance with AGADIR algorithm prediction, the position of the amide I band at 1655 cm-1 in the corresponding FTIR spectrum of P(R) was characteristic of an R-helical structure (Figure 2 A, trace a).41 Low-magnification AFM examination of the air-exposed, hydrophobic side of P(β) monolayers revealed a relatively flat but grainy surface, suggesting a heterogeneous organization of the peptide within the LB film (Figure 3A). High-magnification (500-1000 nm) scans showed that the grainy surface was attributable to the presence of globular aggregates, most often closely packed (Figure 3B). The diameter of aggregates varied between ∼8 and 40 nm, indicating the participation of a variable number of individual peptides in their formation. The aggregates protruded by ∼0.6 nm from a darker matrix. This suggested the coexistence of at least two forms of P(β) at the hydrophilic/hydrophobic interface, one nonaggregated, forming the matrix, and the second corresponding to the aggregates. Unexpected for an R-helical peptide, the surface of P(R) monolayers was decorated by numerous thin (9-25 nm in diameter) and long (up to 1000 nm) filaments (Figure 3C) protruding 0.3-0.4 nm (Figure 3 E) from a rough matrix (Figure 3D), again indicating the presence of at least two different forms of P(R) in the film. (41) Dousseau, F.; Pezolet, M. Biochemistry 1990, 29, 8771.

It is also noteworthy that film structure heterogeneity was not detectable from analysis of the compression isotherms. Mixed Peptide-Phospholipid Films: Low Peptide Molar Fraction. For each peptide, the FTIR spectra obtained for transferred mixed monolayers were almost identical, whatever the nature of the phospholipid and the peptide molar fraction (0.05 or 0.50), although the spectrum corresponding to the low molar fraction was less defined for sensitivity reasons (data not shown). As was done for pure peptide films, all spectra for P(β)phospholipid mixed films were characterized by the presence of an amide I band component centered at 1625 cm-1, which identifies a dominant β-type conformational state (Figure 2B, trace b). For P(R) the R-helical structure was retained under all conditions as revealed by the presence of an amide I band at 1655 cm-1 (Figure 2B, trace a). We then examined the effects of varying the headgroup of phospholipids, in the liquid condensed (LC) state, from zwitterionic (DPPC) to negatively charged (DPPG), on the structure of mixed peptide-phospholipid (1:20 mol/mol) films. Control experiments on pure LC phospholipid films gave flat and uniform surfaces, in accordance with the literature42,43 (data not shown). Images of mixed films at (42) Rinia, H. A.; Demel, R. A.; van der Eerden, J. P. J. M.; de Kruijff, B. Biophys. J. 1999, 77, 1683.

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Figure 5. High-magnification imaging of mixed peptide-saturated phospholipid films at low peptide concentration (5 mol %): (A) P(β)-DPPC; (B) P(R)-DPPC; (C) P(β)-DPPG; (D) P(R)-DPPG. Scan size: 5 µm.

low magnification show that both P(β) (Figure 4A) and P(R) (Figure 4B) markedly modified the structure of the LC DPPC phase. Long elongated LC domains, protruding from a thinner, liquid expanded (LE) matrix were observed for P(β) and P(R) in the presence of DPPC. This strongly suggests a predominant role of dipole-dipole repulsion between DPPC lipid molecules of the LC phase over the line tension at the boundary of LE domains which included the peptides.44,45 Exchanging the PC headgroup for PG had a major influence on the films’ structure. Thus, at this low magnification, the P(β)-DPPG topography was comparable to that of pure P(β) (Figure 4C). On the other hand, large round-shaped LC DPPG domains were observed at the surface of P(R)-DPPG samples (Figure 4D). Decreasing the scan’s size gave more information about peptide-lipid interactions in the mixed films. Closer examination of P(β)-DPPC films showed that, besides the DPPC LC phase (zone 1) and the P(β)-conntaining DPPC LE phase (zone 2) domains, long and thin (∼12 nm in diameter) filaments were present in the LE phase (Figure 5A, arrows). Compared with pure P(β) films, the existence of a smooth LE phase indicated that P(β)-DPPC interaction could ensure miscibility and prevent the strong P(β)-P(β) hydrophobic interactions leading to the formation of globular peptide aggregates. The simultaneous presence (43) Yuan, C.; Johnston, L. J. Biophys. J. 2000, 79, 2768. (44) Keller, D. J.; Korb, J. P.; McConnell, H. M. J. Phys. Chem. 1987, 91, 6417. (45) McConnell, H.M.; Moy, V. T. J. Phys. Chem. 1988, 92, 4520.

of filaments also suggested that DPPC might either induce a privileged orientation of peptides or, as proposed earlier for another primary amphipathic peptide,24 lead to the formation of mixed P(β)-DPPC linear supramolecular structures of unknown stoichiometry. High-resolution images of P(β)-DPPG, with peptide-phospholipid aggregates similar to those of pure P(β) (Figure 5C) confirm that the nature of the polar headgroup played a determinant role in the P(β)-phospholipid mixing properties. High-resolution images of P(R)-DPPC (Figure 5B) also confirmed the presence of a smooth peptide-phospholipidcontaining LE phase (zone 2). In contrast with pure P(R) (and P(β)-DPPC), no filaments were observed in these samples. Small (∼50 nm) circular domains (pure DPPC or P(R)-DPPC) also protrude from the LE phase by a height similar to that of LC domains. The space between large LC DPPG domains was also filled by a small circular domain surrounded by thin layers of LE phase (Figure 5D). Summarizing, P(β)-DPPG was so far the only condition where the apparent structure of the P(β) film was not modified by the presence of the lipid in the mixture. This prompted us to examine the effect of unsaturation on the peptide-lipid interaction. As shown by Figure 6A, a reorganization of the topography occurred with elongated structures, up to 200 nm in length, emerging by ∼0.4 nm from the LE DOPG matrix upon replacement of DPPG by DOPG in P(β)-PG (20:1) films. This indicated that the degree of unsaturation altered the structure of the P(β)-PG aggregates. Changing

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interactions with diunsaturated PC. As shown by Figure 7C, the surface of P(R)-DOPG also consisted of tightly packed filamentous structures, indicating that, at high peptide-to-lipid ratio, the more favorable interaction between P(R) and the unsaturated negatively charged phospholipid did not prevent formation of filaments. In contrast, only a few elongated aggregates decorated the film surface of (1:1) P(R)-DPPG samples (Figure 7D). The topography was characterized by the absence of LC DPPG domains, indicating that most of the DPPG either has directly interacted with P(R) or was affected by the neighboring P(R)-DPPG interactions. On the other hand, LC domains emerging from an LE-like matrix, where a few long filaments seemed to serve as anchors, were observed on P(R)-DPPC films (Figure 7E). This strongly suggested weakening of peptide-lipid interactions when saturated PG was replaced by saturated PC.

Figure 6. Effect of phospholipid unsaturation on peptidephospholipid interaction. Peptide concentration: 5 mol %. Key: (A) P(β)-DOPG; (B) P(R)-DOPG; (C) P(R)-DOPC. Scan size: 1 µm. z scale: 3 nm.

from the β-sheet-forming peptide to the R-helix P(R), the surface of P(R)-DOPG was smooth and practically devoid of clusters or aggregates, suggesting miscibility of the two film constituents (Figure 6B). This miscibility in the LE phase was however dependent upon the phospholipid headgroup as shown by the reappearance of the thin and long P(R) filaments in the P(R)-DOPC (20:1) films (Figure 6C). Mixed Peptide-Phospholipid Films: High Peptide Molar Fraction. Tightly packed long filaments, up to 1 µm in length, also occupied the surface of (1:1) P(β)DOPC samples (Figure 7A). Similar images were obtained for (1:1) P(R)-DOPC samples (Figure 7B). Thus, β-sheet P(β) and R-helical P(R) (see Figure 2B) could give rise to comparable supramolecular filamentous structures upon

Discussion Evidence for the heterogeneous structure of an LB film made of a primary amphipathic peptide CPP was previously reported.23 Like in the present experiments, this occurred for samples transferred well below the collapse pressure, but at a pressure above 25 mN/m, i.e., sufficiently high to prevent film reorganization.39,40 It is worth noting that globular and filamentous complexes were obtained with β-sheet P(β) and R-helical P(R) conformations, respectively. This situation markedly differs from those described for various peptides, in particular amyloid peptides, where the formation of filaments is generally associated with a β-sheet structure.46-49 Observation of similar structures in mixed peptidelipid films strengthened the view that the supramolecular complexes were not a consequence of transfer artifacts. Moreover, as illustrated for P(β)-DOPC and P(R)-DOPG mixtures at high peptide:lipid ratio, the present FTIR and AFM data establish that, according to the lipid used, the primary amphipathic peptides can form filaments despite the β-sheet or R-helical structure. These studies also show that, while maintaining the β-sheet structure, the peptide globular or filamentous organization depends on the phospholipid polar headgroup and acyl chain composition. Thus, the shape of supramolecular complexes is independent of the peptide secondary structure. The composition of globular aggregates and filaments cannot be ascertained from the AFM and FTIR experiments. However, the observation of comparable images for pure P(β) and P(β)-DPPG films at a low peptide:lipid ratio strongly suggests that mixed peptide-lipid complexes were formed. This was supported by the recently obtained evidence for the formation of hydrophobic P(β)phospholipid complexes by electrospray mass spectrometry.50 The diameters of globular structures and filaments were in the range of those previously reported for the other primary amphipatic peptides studied so far.23,24 Due to the AFM “tip effect” and to the use of different tips for each experiment,51 no attempt was done to analyze quantitatively the possible influence of phospholipids on the size of supramolecular complexes. (46) Schladitz, C.; Vieira, E. P.; Hermel., H.; Mo¨hwald, H. Biophys. J. 1999, 77, 3305. (47) Kowalewski, T.; Holtzman, D. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 3688. (48) Gossal, W. S.; Clark, A.H.; Pudney, P. D. A.; Ross-Murphy, S. B. Langmuir 2002, 18, 7174. (49) Fung, S. Y.; Keyes, C.; Duhamel, J.; Chen, P. Biophys. J. 2003, 85, 537. (50) Li, Y.; Heitz, F.; Le Grimellec, C.; Cole, R. B. Rapid Commun. Mass Spectrom. 2004, 18, 135. (51) Engel, A.; Schoenenberger, C.-A.; Mu¨ller, D. J. Curr. Opin. Struct. Biol. 1997, 7, 279.

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Figure 7. AFM topographic images of mixed peptide-phospholipid (1:1) LB films: (A) P(β)-DOPC; (B) P(R)-DOPC; (C) P(R)-DOPG; (D) P(R)-DPPG; (E) P(R)-DPPC. Scan size: 1 µm (A-C); 2 µm (D, E). z scale: 3 nm (A-C); 5 nm (D, E).

Coexistence of LC and LE phases associated with the presence of filaments on P(β)-DPPC (1:20) samples indicates that, like in pure peptide films, the peptide can also exist under different forms in the same mixed peptide-lipid film. Thus, formation of the LE from DPPC at 25 mN/m can be attributed to P(β)-DPPC interactions. The topography of the LE phase is undiscernible from that of a pure lipid LE phase, such as pure DOPC or DOPG. This strongly suggests that the P(β) hydrophobic part is homogeneously dispersed in DPPC. On the other hand, P(β) simultaneously also forms supramolecular complexes as shown by the presence of filaments on the samples, which indicates that the dispersed and filamentous forms are close energetically. The same situation was found for P(R)-DPPC (1:1) samples. Transition from the LC to the LE phase of dipalmitoyl phospholipids helped the detection of the two peptide organizations in the monolayer. The presence of a similar situation in mixed peptideunsaturated phospholipid (1:20) films, undetectable because the lipids are already in the LE phase, cannot be excluded. Molecular self-assembly resulting in aggregation or oligomerization is believed to play a key role in the membrane disruption properties of antimicrobial peptides.10-13 The different models involve a reorientation of at least a part of the concentrated peptides that lie on the surface of the membrane (surface state, S) toward a transbilayer insertion (insertion state, I). The S T I transition depends on the peptide concentration and lipid composition.52 Like many antimicrobial peptides,13 P(β) and P(R) are positively charged and are amphipathic molecules.

Taken together the data suggest that these particular primary amphipathic CPPs may share with antimicrobial peptides, to some extent, common mechanisms of interaction with membranes. Conclusion The presence of either globular or filamentous aggregates in LB films made of pure P(β) or P(R) peptides, and transferred below the collapse pressure, demonstrates the propensity of these primary amphipathic peptides to selfassociate and form supramolecular complexes at a hydrophilic/hydrophobic interface. Similar supramolecular structures could be observed in mixed peptide-phospholipid films. However, their existence and the globular or filamentous shapes were a function of the phospholipid headgroup and acyl chain saturation as well as of the peptide-to-phospholipid molar ratio. Moreover, these structures often coexisted with an LE phase composed of miscible peptide-lipid. The presented data strongly suggested that as for antimicrobial peptides, the primary amphipathic CPP function could be dependent on the local formation of aggregates or oligomers which results from their interaction with membrane phospholipids. Acknowledgment. This work was supported by EU Grant QLK2-CT-2001-01451. LA048622B (52) Heller, W. T.; Waring, A. J.; Lehrer, R. I.; Huang, H. W. Biochemistry 1998, 37, 17331.