Hexagonal Phase Formation in Oriented DPPC–Melittin Samples

CHAPTER SIX

Hexagonal Phase Formation in Oriented DPPC–Melittin Samples: A Small-Angle X-ray Diffraction Study Tanja Pott*,1, Philippe Méléard*

*Univeriste´ Europe´ene de Bretagne, UMR CNRS-ENSCR 6226 “Science Chimique de Rennes”, ENSCR, 11 Alle´e de Beaulieu CS 50837, 35708 Rennes cedex 7, France 1 Corresponding author: e-mail address: [email protected]

Contents 1. Introduction 2. Materials and Methods 3. Results and Discussion 3.1 Characterization of the oriented DPPC–melittin system 3.2 Is the hexagonal phase originating from mismatch? Acknowledgment References

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Abstract We investigated the influence of melittin on the organization of macroscopically oriented dipalmitoylphosphatidylcholine multilayers at 100% relative humidity as a function of temperature and peptide content by small-angle X-ray diffraction. Experiments were done under conditions known to lead to disk formation, a long-lived metastable state, below the transition temperature (Tm), of the pure lipid and in excess water. For T > Tm the system stays in a lamellar organization up to a lipid-to-peptide molar ratio, Ri, of 5, that is, the lowest Ri investigated herein. It was found that the macroscopically oriented system shows a rather complex behavior only below Tm. For T < Tm and for low peptide concentrations (Ri ¼ 200) formation of the rippled phase was found to be abolished. At Ri ¼ 100 melittin induces the formation of a rippled phase at relative low temperature (29  C). At higher peptide content and T < Tm melittin induces the formation of a hexagonal phase, presumably metastable, in coexistence with a lamellar gel phase. A parallel is made with the well-known disk formation. An interpretation in terms of mismatch between the length of the peptide helix and the bilayer thickness is proposed.

Advances in Planar Lipid Bilayers and Liposomes, Volume 20 ISSN 1554-4516 http://dx.doi.org/10.1016/B978-0-12-418698-9.00006-X

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2014 Elsevier Inc. All rights reserved.

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1. INTRODUCTION Many organisms, fungi as well as animals, produce small amphipathic helix-forming peptides that exhibit antibiotic, antifungal, and hemolytic activities (see e.g., [1] and references therein). They act by perturbing the barrier function of the membrane, leading to cytolysis and death of cells [2–5]. The amphipathic and basic peptide melittin from the European honeybee (Apis mellifera) venom is one of those (for review, see [6–8]). Much attention has been paid to its particular strong lytic potency on natural and artificial membranes [9–13] and its ability to form voltage-dependent ion channels [14–16]. Melittin is further known for its capacity to induce morphological changes in lipid bilayers [17]. Especially well characterized is its action on phosphatidylcholine bilayers where the peptide induces a reversible disk-to-vesicles transition (at lipid-to-peptide molar ratios, Ri  20), triggered by the melting of the fatty acyl chains [18–21]. In the fluid phase, it induces the formation of large unilamellar vesicles, LUV, with a diameter of about 4000 A˚, whereas in the gel phase disks with a diameter ˚ and a thickness of a single bilayer are found [18,19]. It has been of 200-400 A reported earlier that the system has to be incubated above the main transition temperature (Tm) to show this reversible disk-to-vesicles transition [18,19], but it has later been shown that for dipalmitoylphosphatidylcholine (DPPC) membranes the incubation above the pretransition temperature (Tp) is sufficient to induce disk formation [22–24]. In the Lb0 -phase, these disks are metastable and fuse to yield large aggregates on the timescale of hours to days depending on the lipid [21,25,26]. For very high peptide amounts, Ri  5, the disk-to-vesicle transition disappears and so-called mixed micelles are observed over the entire temperature range. Numerous studies addressed the question how the peptide helix is oriented in the lipid bilayer. From indirect measurements, it has been suggested that the orientation of melittin in the phosphatidylcholine bilayers changes from parallel to perpendicular to the membrane plane when passing from the fluid to the gel phase [17,25]. A perpendicular orientation of melittin has also been proposed in the case of cholesterol-containing discoidal objects [23]. However, direct investigations of the peptide orientation in a lipid bilayer are most conveniently studied using macroscopic oriented samples. Amazingly, the peptide-induced polymorphism in excess water does not prevent the formation of highly oriented lipid–peptide complexes even at low Ri (see for instance [16,27–30]). From such studies on

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phosphatidylcholine–melittin systems, transbilayer models [16,31,32] as well as wedge-like models [33,34] have been proposed. The peptide orientation appears further to be sensitive to the hydration of the bilayer, the lipid composition, and its physical state [31,32,35–37]. It has also been pointed out that the way the oriented samples are prepared might influence the peptide orientation [16]. Melittin has further been shown to insert into the membrane in a concentration-dependent manner, with a well-defined transition from S-to-I state [38]. The “surface” S-state corresponds to peptides that are oriented parallel to the plane of the membrane, cause membrane thinning and do not form pores. The “inserted” I-state corresponds to peptides oriented perpendicular to the membrane plane that form nontransient pores, whereas the bilayer thickness remains constant [29,38–42]. In the case of oriented melittin–phosphatidylcholine systems, data obtained by Huang and coworkers is by far the most consistent. Anyhow, melittin is known to induce metastable states that are important for understanding the action of melittin on membranes. Long-lived metastable states may further explain controversial results. Herein, we investigated the behavior of macroscopically oriented DPPC samples at 100% relative humidity (RH) containing various amounts of melittin as a function of temperature by small-angle X-ray diffraction under conditions known to lead to disk formation, thus to a long-lived metastable state. We will show in the following that the system is characterized by large regions where phase coexistence occurs. The most remarkable feature of this system is the observation of a hexagonal lattice coexisting with a lamellar one at T  Tm and in a large range of peptide contents. A single lamellar phase is only detected at high temperature, that is, T  Tm.

2. MATERIALS AND METHODS Synthetic DPPC was obtained from Avanti Polar Lipids Inc. (Birmingham, AL). Highly purified melittin was purchased from Serva (Heidelberg, Germany) and used without further purification. Oriented samples were prepared from the aqueous phase rather than from organic solvent because it cannot be ruled out that passing through the organic phases induce lipid–peptide assemblies that are different from those prepared in excess water. Samples were thus prepared in excess water and submitted to freeze-thaw cycles until homogeneous dispersions were obtained. Melittin containing samples were further incubated at 60  C. Typical phenomena occurring in these systems, such as the disk-vesicles transition at

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Ri ¼ 20 or the formation of mixed micelles at Ri ¼ 5 [18,19], could indeed be observed in these samples. Oriented samples were then obtained by pipetting aqueous lipid dispersion on a 75-mm thick curved glass plate followed by dehydration. The procedure was repeated until 1.5 mg lipid was deposed on a 6 mm  6 mm surface, after which the samples were equilibrated at 100% RH. To improve macroscopic orientation the samples were cycled around the phospholipid main transition. This preparation produces a stack of 1500-2000 highly oriented (mosaic spread <5 ) bilayers. The experiments were carried out in the laboratory using a rotating Cu anode X-ray generator (Bruker Nonius FR59) equipped with a 2D reflection system (Montel 200 multilayer graded optic), used as a monochromator ˚ ). Three sets of vertical and and collimator (Ka wavelength l ¼ 1.54187 A horizontal slits were used, leading to a spot size of 0.5 mm  0.5 mm. A 2D Marresearch imaging plate detector (mar345dtb) with a plate diameter of 345 mm and a pixel size of 150 mm  150 mm was used to collect the data. The sample to film distance was 825 mm (resolution 0.01 A˚1). The spot size, defined by three sets of vertical and horizontal slits, was approximately 1 mm  1 mm. The sample holder was equipped with humidity chambers containing pure water to equilibrate the sample with the air of defined RH (100%) and allowing precise temperature adjustment ( 0.1  C). Temperature variation was done by decreasing the temperature stepwise from 57.3 to 29  C. For a given temperature, diffraction patterns were recorded once Bragg reflection stayed stable for at least 1 h. Samples were subjected to thin layer chromatography after the X-ray measurements to prove phospholipid chemical integrity. It should also be noted that such a standard humidity chamber does not allow full lipid multilayer hydration even not at 100% RH, but always stays somewhat lower. This is also called the vapor pressure paradox [43].

3. RESULTS AND DISCUSSION 3.1. Characterization of the oriented DPPC–melittin system Small-angle X-ray diffraction experiments have been performed at Ri ¼ 1, 200, 100, 50, 30, 20, 15, 10, and 5 and 100% RH in the temperature range from 57.3 to 29  C (in approximately 5  C steps), that is under conditions where in excess water the vesicle to metastable disk transition occurs. Indeed, Lb0 -phase DPPC–melittin disks are metastable and disappear only gradually after days [21]. In Fig. 6.1, 2D diffraction patterns of the DPPC

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A

B

C

D

E

F

Figure 6.1 2D diffraction pattern of the DPPC–melittin system at 29  C, 100% RH and (A) Ri ¼ 1, (B) Ri ¼ 200, (C) Ri ¼ 100, (D) Ri ¼ 50, (E) Ri ¼ 10, and (F) Ri ¼ 5. It should be noted that equatorial reflections, that is, those at qz ¼ 0, are attenuated due to the diffraction geometry.

system at 29  C and 100% RH containing various amounts of melittin are presented. As a single lamellar phase is observed for T > Tm and whatever the melittin content, the corresponding diffraction patterns are not presented. The diffraction pattern of pure DPPC shows the intense reflections (h ¼ 1–6) of a lamellar organization with a repeat distance of 59.7 A˚ (Fig. 6.1A), characteristic for the Lb0 -phase [44,45]. A trace of a second phase (actually Pb0 ) can also be detected that gives rise to weak reflections in the direction of the bilayer normal qz and symmetric satellites close to this axis.

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This is typical for the Pb0 or so-called rippled phase, which consists of an oblique unit cell, leading hence to a 2D diffraction pattern [46,47]. Schematics of rippled bilayers in the Pb0 -phase and the corresponding diffraction pattern are shown in Fig. 6.2B. As these samples are oriented only in the direction perpendicular to the glass plate but not parallel to the support, we actually deal with a cylindrical distribution of domains. The reciprocal space structure of such a cylindrical powder is thus obtained by rotating Fig. 6.2B around the qz-axis, leading to symmetrization of the diffraction pattern. The unit cell of the small amount of Pb0 -phase present in the DPPC ˚ , b ¼ 155 A ˚ , and g  96 , system at 29  C is characterized by a ¼ 61.5 A which is very similar to values reported in literature [46]. However, the structural parameters of the DPPC Pb0 -phase are strongly dependent on the precise experimental conditions (hydration, heating, cooling, for details see [45] and references therein). It should therefore be noticed that the system herein is not fully hydrated due to the vapor pressure paradox [43] and that the Pb0 -phase diffraction is obtained by slow, but stepwise cooling.

Figure 6.2 Schematic presentation of the different lattices present in our samples and their corresponding small-angle diffraction pattern. (A) One-dimensional lamellar lattice, (B) two-dimensional oblique unit cell, typical for a so-called rippled phase, and (C) two-dimensional hexagonal array.

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The incorporation of very small amounts of melittin (Ri ¼ 200) results in a marked change in the diffraction pattern at 29  C (Fig. 6.1B). The coexistence of several phases can be recognized. Two lamellar phases with similar repeat distances of d ¼ 60.6 and 60.0 A˚ (the latter being predominant) are more easily seen for the higher order reflections where their slight difference in d becomes better resolved. Apart from these two lamellar matrices, there are three more reflections at qz ¼ 0.118, 0.218, and 0.235 (see arrows Fig. 6.1B). From the evolution of the diffraction patterns with temperature one may tentatively index the weak reflection at qz ¼ 0.118 and 0.235 as h ¼ 1 and 2 of a lamellar phase. The corresponding repeat distance of ˚ may indicate a fluid-like organization. Indeed, examination of the 53.4 A diffraction patterns as a function of temperature shows that these reflections originate from the fluid phase. The faint reflection at qz ¼ 0.218 remains unassigned. Also the Ri ¼ 200 sample does not show any satellites in the q? direction whatever the temperature, demonstrating the disappearance of the Pb0 -phase. It should be noted that the inhomogeneity of this sample at T < Tm is not due to short-lived metastable states, but remains constant in reciprocal distance as well as intensities for at least some hours. At Ri ¼ 100 and 29  C the sample gives rise to a 2D diffraction pattern with pronounced satellites near the meridian (Fig. 6.1C). The pattern is characteristic of asymmetric ripples (see also Fig. 6.2B), but in contrast to the pure lipid this rippled phase dominates the diffraction pattern well below Tp of DPPC. From this pattern, we find a ¼ 61.2 A˚, b ¼ 164 A˚, and g  105 , b being thus longer and g greater than in the Pb0 -phase of pure DPPC observed herein, thus showing peptide-triggered formation of asymmetric ripples. Apart from the reflections of the rippled phase one additional weak reflection at qz ¼ 0.137 is detected (see arrow in Fig. 6.1C). Similar to the system at Ri ¼ 200, the evolution of the reflection with temperature indicates that this reflection is due to small amounts of remaining fluid phase ˚ . Again, reciprocal distances as well as intensities with a periodicity of 45.8 A stays constant for at least some hours, just like the metastable melittininduced disks formed in excess water at the same temperature. A 2D diffraction pattern is also observed at Ri ¼ 50 (Fig. 6.1D), but with features completely different from the Ri ¼ 100. Actually, this image consists of a superposition of a lamellar and a hexagonal pattern. One further notes that the lamellar phase exhibits no longer a ripple structure (the lamellar reflections in Fig. 6.1D are labeled with consecutive numbers). The 2D hexagonal phase is presumably a cylindrical powder, similar to a rippled phase, but as its reciprocal space structure is symmetric the rotation around qz does

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not change the appearance of the diffraction pattern (see Fig. 6.2C). It is noteworthy that the hexagonal reflections occur along the qz-axis and not along the in-plane axis, in contrast to dried purple membranes, where the transmembrane a-helices of bacteriorhodopsin give rise to a hexagonal in-plane array [48–50]. Herein, the hexagonally packed cylinders have their long axis parallel to the membrane plane of the coexisting lamellar phase. ˚ , corresponding The hexagonal lattice at Ri ¼ 50 has a periodicity of 49.1 A to a center-to-center separation of the hexagonal cylinders of 56.7 A˚. The coexistence of a lamellar and a hexagonal phase can easily be observed up to Ri ¼ 10 (see arrows Fig. 6.1E). A similar coexistence has been mentioned briefly for an oriented DMPC-melittin system [51]. Anyhow, examination of the diffraction images reveals a decrease in the total intensity of reflections with increasing peptide content. This may be indicative of domain formation with no or very-low stacking order. At the highest melittin content studied herein, Ri ¼ 5, the diffraction pattern shows mainly 1D organization (Fig. 6.1F). The reflections can be indexed as a lamellar phase with a repeat ˚ . Two further reflections of weak intensity at qz ¼ 0.155 and distance of 63 A 0.266 are observed (see arrows in Fig. 6.1F). In spite of the fact that reflections beside those on the qz-axis are not obvious it might be suggested that the hexagonal phase is still present in the system. Anyhow, it is interesting to compare this most probably metastable hexagonal phase with what occurs in excess water. In excess water and in the same range of Ri, melittin transforms the complete DPPC gel phase into small metastable disks where the peptide is tough to be located at the edge only [17,21]. This melittin organization would exert strong mechanical constraints only on a fraction of the lipids. DPPC molecules located far away from the disks edges would not be strongly perturbed. Figure 6.3 shows the variations in the repeat distances as a function of melittin content at 29 and 57.3  C, respectively. It has been pointed out that phospholipids are extremely sensitive to humidity, especially near 100% RH [52]. Slight changes in the humidity can induce variations in the ˚ according to Wu and coworkers. d-spacing of multilayers as much as 1 A However, strong variations in the repeat distance can usually be interpreted as due to variations in the bilayer thickness [52]. In the La-phase of DPPC increasing melittin concentration leads to a decrease in the repeat distance (Fig. 6.3A). This decrease in d appears not to be continuous and one may ˚ and it is define two regions. For Ri 20 the decrease is smaller than 1 A questionable if this behavior reflects a decrease in the bilayer thickness. For lower Ri the decrease in d largely exceeds 1 A˚ and is most probably

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Repeat distance (Å)

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A

B

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C

0

0.05

0.1

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Figure 6.3 Variations of the d-spacing in the DPPC–melittin systems as a function of melittin content at 100% RH, (A) at 57.3  C, (B) for the predominant lamellar phase at 29  C, and (C) of the hexagonal lattice at 29  C. The center-to-center separation of the hexagonal cylinders, a, is related to the d-spacing by a ¼ (2/√3)d10.

related to a decrease in bilayer thickness. In general, at low concentration amphipathic peptides seem to be adsorbed at the membranes surface leading to membrane thinning, whereas high concentrations result in insertion of the peptide and a sudden change in the evolution of the bilayer thickness [29,38,52–54]. Our data might be consistent with the idea of melittin being in the S-state in the DPPC fluid phase for whatever the Ri. Anyhow, further investigations and determination of the bilayer thickness are clearly necessary to understand the observation of a continuous decrease in d up to Ri ¼ 5. At 29  C the d-spacing of the predominant lamellar phase shows a different evolution as a function of peptide content than at 57.3  C (Fig. 6.3B). For low amount of melittin (Ri ¼ 200 and 100) d is almost the same as for pure DPPC. At Ri ¼ 50 the repeat distance increases suddenly of about ˚ . For lower Ri the d-spacing shows small variations but no apparent 3A

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systematic evolution. Interestingly, the sudden increase in d at Ri ¼ 50 and 29  C coincides with the appearance of the hexagonal lattice. The periodicity of the hexagonal lattice at 29  C decreases with increasing peptide amount up to Ri ¼ 15 (Fig. 6.3C). At Ri ¼ 10 a reincrease is observed. A change in the composition of the cylinder would account for such a variation. Interestingly the d-spacing of the coexisting lamellar phase remains distinctly higher than that of pure DPPC (Fig. 6.3B). A reason for that might be a better overall hydration of the gel phase lipids due to the peptide presence in the samples. Indeed d-spacings around 63 A˚ are much closer to the 63.7 A˚ reported in [55] or the 63.2 A˚ in [56]. As the phase behavior of the DPPC–melittin system at 100% RH appears to be rather complex a diagram depicting the different observed states is shown in Fig. 6.4. Even at very small amounts (Ri ¼ 200) of melittin added to DPPC one observes the appearance of a fluid-like lamellar phase at T < Tm coexisting with gel phases (the open squares in Fig. 6.4). The fluid-like phase disappears when the hexagonal arrangement becomes visible. It seems that the region of coexistence extends towards higher temperature with increasing melittin content. This is in agreement with earlier

60

Temperature (⬚C)

55

Fluid

La

50 Fluid + gel

45 40 35

Lb ⬘ +

30

Pb ⬘ •

Rippled + Fluid

Gel + hexagonal

100

10 Ri

Figure 6.4 Phase behavior of the DPPC–melittin system at 100% RH as deduced from small-angle X-ray measurements, that is under conditions where long-lived metastable states are observed: ○, lamellar fluid phase; ▪, lamellar gel phase; □, coexistence of lamellar gel and fluid phases; ▲, coexistence of rippled and fluid phase; ⧫, coexistence of lamellar gel and hexagonal phase; , coexistence of lamellar gel and fluid phase and hexagonal lattice. The presentation of the x-axis was chosen as to help reading the figure.

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reports of a melittin-induced decrease in the cooperativity of the gel-to-fluid transition [26,57]. At low Ri the transition in DPPC–melittin complexes was described as broad and biphasic [26] and similar conclusions have been drawn from a study with high Ri [57]. However, from our data it is clear that a deduction of a preferred orientation of melittin within DPPC bilayers in the low temperature range becomes a difficult task.

3.2. Is the hexagonal phase originating from mismatch? It should be noted that the hexagonal phase found herein is induced by a lowering of the temperature at fixed Ri quite in contrast to hexagonal phase forming lipids (e.g., phosphatidylethanolamines). Further it seems to be rather surprising that this hexagonal phase is found at T < Tm. In Fig. 6.5 only the first-order reflections of the lamellar and the hexagonal phases at Ri ¼ 20 are shown as a function of the temperature. When lowering the temperature it can be seen that the lamellar fluid phase reflection (h ¼ 1) decreases continuously in intensity down to 39  C where the hexagonal phase reflection (h ¼ 1, k ¼ 0) appears superposed on the lamellar diffraction. At T < 39  C only the hexagonal reflection remains in this q-range. This indeed indicates that this metastable hexagonal phase originates from the melittininduced lamellar fluid phase (see also Fig. 6.4). Even though the nature of the hexagonal phase (inverse or direct) cannot be deduced from this study, the latter observation is a strong indication in favor of an inverted hexagonal phase. Indeed, the model given below assumes an inverse type structure. How can the formation of a hexagonal phase be understood? Quite a large number of peptide-induced hexagonal phases have been reported. Especially notable are the inverted hexagonal phases that are formed by transmembrane peptides through mismatch in their length with the lipid hydrophobic thickness [58–60]. In this case, the peptide is thought to occupy the axis-to-axis plane between two neighboring cylinders. It might be possible that such a situation is also encountered in the DPPC–melittin system at T < Tm as discussed in the following. Mismatch has already been invoked in the context of the melittininduced disks [21]. So let us begin with the phase behavior of melittin– DPPC in excess water. Disk formation occurs in the range 100 > Ri > 5 [19] where we also observe the hexagonal phase. These morphological changes appear thus to be related to each other. In the disks, melittin surrounds the hydrophobic borders in a pseudotransmembrane manner [17,21,23,61] with an interhelical angle of 120 in the case of gel phase DPPC [62]. Further the

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Figure 6.5 The first-order reflections of the lamellar phase (h ¼ 1) and the hexagonal phase (h ¼ 1, k ¼ 0) of the DPPC–melittin system at Ri ¼ 20 are shown as a function of the temperature. Temperatures are denoted on the right side of the profiles ( C) and intensities are log-scaled.

disks are metastable at T  Tm and fuse with time to yield large aggregates [21,25,26]. This metastability has been proposed to be the result of a mismatch between the peptides a-helix length and the bilayer thickness [21]. In our study, the disks formed in excess water were fused together by the process of macroscopic orientation of the sample and the accompanying reduction in water content. Under the hypothesis that a quasitransmembrane orientation of (at least some) melittin molecules is retained, one may imagine that a hexagonal phase is formed as a result of mismatch, similar to what has been reported for gramicidin A [58,59] and hydrophobic a-helical model peptides [60]. In this context, it is interesting to refer to data obtained on fused DPPC– melittin disks in excess water, where an increased number of gauche rotamers compared to pure DPPC has even been found in such a system [26]. Again, it

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has to be mentioned that an average of strongly perturbed and almost unperturbed lipids can explain these results. In excess water the fusion of disks may produce inverted fluid type fusion intermediates in which the lipid chains are highly disordered. In the oriented samples studied herein such intermediates may then arrange in a fluid phase 2D hexagonal array. In conclusion, the study sheds some light on the complex interaction between melittin and gel phase DPPC bilayers due to long-lived metastable states, such as the well-known disks or the mismatch-triggered HII-phase found herein. It is quite remarkable how strongly melittin perturbs some lipid molecules, especially those in close contact with the peptide (HIIphase, edges in disks), whereas others remain almost unperturbed (lamellar gel phase, inner part of the disks). This underlines the importance of local perturbations and constraints exerted by melittin on nearest neighbor phospholipids that might play an important role in its biological action.

ACKNOWLEDGMENT We would like to thank John H. W. Clark for his help in the English proofreading.

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