A study on the interactions of surfactin with …detailed study on the interaction of surfactin with phospholipid vesicles, as monitored by two powerful physical techniques such as

A study on the interactions of surfactin with phospholipid vesicles

Alicia Grau a, Juan C. Gomez Fernandez a, Franc,oise Peypoux b, Antonio Ortiz a;*a Departamento de Bioqu|mica y Biolog|a Molecular-A, Facultad de Veterinaria, Universidad de Murcia, E-30100 Espinardo, Murcia, Spain

b Biochimie Analytique and Synthe©se Bioorganique, Universite Claude Bernard, Lyon 1, Villeurbanne Cedex, France

Received 11 January 1999; received in revised form 25 February 1999; accepted 2 March 1999

Abstract

Surfactin, an acidic lipopeptide produced by various strains of Bacillus subtilis, behaves as a very powerful biosurfactantand posses several other interesting biological activities. By means of differential scanning calorimetry and X-ray diffractionthe effect of surfactin on the phase transition properties of bilayers composed of different phospholipids, including lipidsforming hexagonal-HII phases, has been studied. The interactions of surfactin with phosphatidylcholine andphosphatidylglycerol seem to be optimal in the case of myristoyl acyl chains, which have a similar length to the surfactinhydrocarbon tail. Data are shown that support formation of complexes of surfactin with phospholipids. The ionized form ofsurfactin seems to be more deeply inserted into negatively charged bilayers when Ca2� is present, also supporting theformation of surfactin^Ca2� complexes. In mixtures with dielaidoylphosphatidylethanolamine, a hexagonal-HII phaseforming lipid, surfactin displays a bilayer stabilizing effect. Our results are compatible with the marked amphiphilic nature ofsurfactin and may contribute to explain some of its interesting biological actions; for instance the formation of ion-conducting pores in membranes. ß 1999 Elsevier Science B.V. All rights reserved.

Keywords: Di¡erential scanning calorimetry; X-ray di¡raction; Phospholipid vesicle; Surfactin

1. Introduction

Surfactin, an acidic lipopeptide produced by vari-ous strains of Bacillus subtilis, posses a cyclic struc-ture, containing seven amino acid residues, which isclosed by a C14^C15 lactone ring [1] (Fig. 1).

Surfactin behaves as a very powerful biosurfactant[2], which is an expected behaviour given the amphi-

philic nature of the lipopeptide, with a polar aminoacid head and a hydrocarbon chain (Fig. 1). Besidesits properties as a biosurfactant, surfactin exhibitsseveral other important biological activities: it is anantibiotic substance with antitumour activity [3], itinhibits formation of ¢brin clots [4], it is able tolyse erythrocytes [5] and posses antiviral activity[6,7].

Given its amphiphilic character it is presumed thatthe above-mentioned activities are a direct conse-quence of the interaction of surfactin with its targetmembrane and the modulation of bilayer properties.Thus, and because of all these important biologicalactivities, surfactin has given rise to a great interestfrom scientists, with a considerably growing researchon this topic. In this respect, several variants of sur-

0005-2736 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved.PII: S 0 0 0 5 - 2 7 3 6 ( 9 9 ) 0 0 0 3 9 - 5

Abbreviations: DEPE, dielaidoylphosphatidylethanolamine;DMPC, dimyristoylphosphatidylcholine; DMPG, dimyristoyl-phosphatidylglycerol ; DMPS, dimyristoylphosphatidylserine;DPPC, dipalmitoylphosphatidylcholine; DSPC, distearoylphos-phatidylcholine; DSC, di¡erential scanning calorimetry

* Corresponding author;E-mail : [email protected]

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Biochimica et Biophysica Acta 1418 (1999) 307^319

factin, di¡ering in the peptide backbone have beenisolated [6,8^11]. Furthermore, several laboratoriesare presently applying genetic engineering techniquesto improve surfactin production [12,13].

Despite the growing interest on the possible appli-cations of surfactin in several areas, few works haveattempted to carry out basic studies on the interac-tion of the lipopeptide with phospholipid bilayers. Ina previous work [14], the interaction of surfactin withlipid vesicles and monolayers was studied. By apply-ing circular dichroism spectroscopy and adsorptionexperiments in monolayers, it was concluded that thelipid composition of the monolayer is essential toallow surfactin penetration, and the important roleof calcium was also evaluated.

Here we have extended this work and carried out adetailed study on the interaction of surfactin withphospholipid vesicles, as monitored by two powerfulphysical techniques such as di¡erential scanning cal-orimetry (DSC) and X-ray di¡raction. By means ofDSC, the e¡ect of surfactin on the phase transitionproperties of bilayers composed of di¡erent phos-pholipids, including lipids forming hexagonal-HII

phases, was studied. Small-angle X-ray di¡ractionallowed us to characterize the membrane structurespresent under every particular condition and to de-termine the e¡ect of the incorporation of surfactin onbilayer d-spacing and HII tubules size. The results arediscussed on the light of the e¡ect of bilayer phospho-lipid composition on surfactin penetration and thepossible association with some phospholipid species.

2. Materials and methods

2.1. Materials

Surfactin was produced by Bacillus subtilis S499and isolated and puri¢ed as previously described

[8]. L-K-Dimyristoylphosphatidylcholine (DMPC);L-K-dimyristoylphosphatidylglycerol (DMPG); L-K-dipalmitoylphosphatidylcholine (DPPC); L-K-distear-oylphosphatidylcholine (DSPC), L-K-dimyristoyl-phosphatidylserine (DMPS) and L-K-dielaidoylphos-phatidylethanolamine (DEPE) were from AvantiPolar Lipids (Birmingham, AL, USA). Bicinchoninicacid reagent was purchased from Pierce, Rockford,IL, USA. All the other reagents were of the highestpurity available. Water was twice-distilled in an all-glass apparatus and deionized using a Milli-Q equip-ment from Millipore.

Stock solutions of surfactin and the various phos-pholipids were prepared in chloroform/methanol(2:1) and stored at 320³C.

2.2. Di¡erential scanning calorimetry

Samples for DSC were prepared by mixing theappropriate amounts of phospholipid (usually 3Wmol) and surfactin (as indicated) in chloroform/methanol (2:1). The solvent was gently evaporatedunder a stream of dry N2, to obtain a thin ¢lm atthe bottom of a small thick-walled glass tube. Lasttraces of solvent were removed by a further 2 h des-iccation under high vacuum. To the dry samples, 40Wl of a bu¡er containing 150 mM NaCl, 0.5 mMEDTA, 20 mM Tris (pH 7.4) was added, and vesicleswere formed by vortexing the mixture, always keep-ing the temperature above the highest gel to liquid^crystalline phase transition of the sample. In someexperiments using DMPG to investigate the e¡ectof surfactin on a negatively charged membrane inthe absence and presence of calcium, 150 mMNaCl, 20 mM Tris (pH 8.5) bu¡er, containing di¡er-ent calcium concentrations (as indicated) was used.

Approximately 25 Wl of the vesicle suspensions wassealed in small aluminium calorimetry pans andscanned. Scans were carried out in a Perkin^ElmerDSC-7 apparatus, at heating and cooling rates of4³C min31. The calorimeter was calibrated using in-dium as standard.

Partial phase diagrams for the phospholipid com-ponent were constructed as previously described [15].The solidus and £uidus lines of the diagrams werede¢ned by the onset temperature of the transitionpeaks obtained from heating and cooling scans, re-spectively. In order to avoid artefacts due to the

Fig. 1. Primary structure of surfactin (n = 9^11).

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thermal history of the sample, particularly on coolingscans, the ¢rst scan was never considered. Secondand further scans were carried out until a reprodu-cible and reversible pattern was obtained, what usu-ally occurred already with the second scan. Further-more, phase diagrams were also constructed takingthe onset and completion temperatures only on heat-ing scans, and the results obtained were exactly thesame.

2.3. Small-angle X-ray di¡raction

Samples for X-ray di¡raction analysis were pre-pared essentially as described above for DSC withsome minor modi¢cations. Usually 10^15 mg of thecorresponding phospholipid was used, and the dry¢lms were resuspended in 1 ml of the same bu¡er.The vesicle suspensions were pelleted down by cen-trifugation in an Eppendorf bench microfuge andplaced in the X-ray di¡ractometer holder.

Nickel-¢ltered Cu KK (V= 1.54 Aî ) X-ray was ob-tained from a Philips (model PW1830) anode. X-rayswere focused using a £at gold-plated mirror and re-corded using a linear position sensitive detector mod-el 210 (Bio-Logic, France). Unoriented lipid disper-sions, prepared as described above, were measured inaluminium holders with Mylar windows. The sampletemperature was kept ( þ 0.5³C) using a circulatingwater bath. The system was allowed to equilibratefor about 5 min at each temperature before measure-ments. Typical X-ray exposure times were 10^15 minfor each sample.

For the measurement of lattice spacing, crystallinecholesterol was used as standard. This compoundshows a sharp re£ection corresponding to a 33.6 Aî

spacing, which is very adequate for calibration. Therelative position of the peaks in the di¡ractogramallows speci¢cation of the packing symmetry of thephase (lamellar, L or hexagonal-HII, HII) and thedistance of the peaks to the centre (non-di¡ractedbeam) allows calculation of the repeat or d-spacingof that particular phase.

2.4. Analytical assays

Phospholipid phosphorous was determined ac-cording to the method of Bo«ttcher [16].

Association of surfactin with DMPC, DPPC and

DSPC bilayers was measured as follows. Multilamel-lar vesicles containing 9 mol% of surfactin in thedi¡erent phosphatidylcholines mentioned above,were prepared as described for DSC. Vesicles werepelleted down in a bench microfuge and surfactinwas determined in the supernatants. Surfactin con-centration was measured by the bicinchoninic acidassay [17], using an aqueous solution of the samelipopeptide as standard. Incorporation of surfactininto DMPC, DPPC or DSPC bilayers was found tobe 97 þ 3%, irrespectively of the type of lipid used,and for all the surfactin concentrations used.

3. Results

3.1. Interaction of surfactin with phosphatidylcholinemembranes

In order the check that the structure of surfactinwas not modi¢ed in the same range of temperatureused for the lipid samples studied, aqueous disper-sions of the lipopeptide were subjected to DSC andno endothermic or exothermic transitions whatsoeverwere detected (not shown). Furthermore, the factthat all the thermograms obtained for the di¡erentlipid^surfactin mixtures were totally reversible andthat successive scans were always identical to the ¢rstscan, fully support that surfactin structure is not al-tered by the temperature scannings carried out in thiswork.

Fig. 2 shows the DSC pro¢les of mixtures of sur-factin with three phosphatidylcholines di¡ering inhydrocarbon chain length, namely DMPC (Fig.2A), DPPC (Fig. 2B) and DSPC (Fig. 2C). Increas-ing the surfactin concentration in DMPC (Fig. 2A)progressively makes the pretransition to decrease,which is completely abolished at 0.5 mol%. The Tc

of the main gel to liquid^crystalline phase transitionis also decreased as the concentration of surfactin isincreased. The Tc of the main transition in coolingscans (not shown) is considerably shifted to highervalues up to a surfactin concentration around 1 mol%and then remains essentially constant. At 1 mol% ofsurfactin, a second peak above the main transitionstarts to be apparent and becomes more prominentas the concentration of the peptide is increased. At asurfactin concentration of 4.7 mol% and higher, this

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A. Grau et al. / Biochimica et Biophysica Acta 1418 (1999) 307^319 309

second peak is even more prominent than the lowerone.

The pattern observed for DPPC bilayers (Fig. 2B)is qualitatively similar, with the di¡erence that the Tc

of the main transition on cooling scans is not in-creased by addition of surfactin. Furthermore, twonew transitions are observed, in this case at 4.7 mol%surfactin, but at 9.1 mol% these are still less impor-tant than the main one.

Finally, the perturbations induced by addition ofsurfactin to DSPC bilayers (Fig. 2C) were muchweaker than in DMPC or DPPC membranes, andno new transitions were observed even at the highestconcentrations used.

From the calorimetric data obtained from thescans shown in Fig. 2, partial phase diagrams wereconstructed (Fig. 3). For the sake of simplicity thepretransitions have been omitted from the phase dia-

Fig. 2. DSC heating-scan thermograms for mixtures of surfactin with DMPC (A), DPPC (B) and DSPC (C). Each sample contained3 Wmol phospholipid and the appropriate amount of surfactin (mol% of total), which is expressed on the curves.

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grams. At ¢rst sight, it is clearly seen that the mix-ture surfactin/DMPC displays a di¡erent behaviourthan with the other PC species. The partial phasediagrams for DPPC and DSPC (Fig. 3B,C) indicatea near-ideal behaviour, with both the solidus and£uidus lines decreasing as the concentration of sur-factin is increased. The diagram for DMPC (Fig. 3A)shows the presence of a £uid-phase immiscibilityfrom surfactin concentrations of 1 mol% and higher.This immiscibility is de¢ned by the Tc (obtainedfrom cooling scans) of the second peak observed inthe DSC experiment (Fig. 2A). In the region de¢nedby G+F at least two, but maybe more, gel and £uidphases should coexist, since two, but maybe more,peaks are observed in the thermograms at surfactinconcentrations above 1 mol% (Fig. 2A).

Fig. 4 shows that vH of the main gel to liquid^crystalline phase transition of the three PC speciesremains essentially constant (within experimental er-ror) upon increasing surfactin concentration.

DSC experiments were also carried out at pH 5.5and 8.5, in order to check the e¡ect of the ionizationstate of the surfactin molecule, and the results wereessentially the same as those described above at pH7.4 (not shown). On the other hand, addition of2 mM Ca2� to surfactin/DMPC mixtures at pH 7.4resulted in the same pattern as that obtained in theabsence of this cation (not shown).

Fig. 3. Partial phase diagrams for mixtures of surfactin withDMPC (A), DPPC (B) and DSPC (C). Filled symbols corre-spond to the solidus line and open symbols to the £uidus line.G corresponds to a lamellar gel phase, and F and FP to lamel-lar £uid phases (see text for explanation). Dashed lines separatedi¡erent regions on the diagram. Data points correspond to themean þ S.E. of three di¡erent experiments. Error bars areshown when bigger than the symbols.

Fig. 4. Dependence of the enthalpy change of the gel to liquid^crystalline phase transition of mixtures of surfactin with DMPC(squares), DPPC (¢lled circles) and DSPC (open circles) on sur-factin concentration. Data points correspond to the mean þ S.E.of three di¡erent experiments. Error bars are shown when big-ger than the symbols.

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The e¡ect of incorporation of surfactin on thephase adopted by the membrane and its properties,was studied in mixtures of surfactin with DMPC, forwhich a stronger interaction was observed by DSC,by performing small-angle X-ray di¡raction experi-ments at di¡erent temperatures (Fig. 5). Interest-ingly, concentrations of surfactin as low as 1 mol%considerably increase d-spacing, particularly whenthe membrane is in the gel state. Thus, at 10³C,i.e., below the phospholipid pretransition, additionof 2 mol% of surfactin increased d-spacing from67.2 Aî to 77.4 Aî , about a 10 Aî increase. This in-crease of d-spacing is still signi¢cant at 20³C, i.e.,between the pretransition and the main transition(ca. 5 Aî ) and at 35³C, in the £uid phase, with abouta 4 Aî increase. In all cases, the re£ections obtainedappeared at a ratio of the lattice parameter of1:1/2:1/3:T, indicating that the systems were alwaysin the lamellar state. This behaviour was essentiallythe same at pH 7.4 and 8.5.

3.2. Interaction of surfactin withdimyristoylphosphatidylglycerol: e¡ect of Ca2+

The DSC pro¢les of mixtures of surfactin withDMPG membranes at pH 7.4, at which the peptide

is essentially protonated, are shown in Fig. 6. A sim-ilar pattern as that shown above for DMPC wasobtained, the pretransition disappearing, the Tc ofthe main transition shifting to lower temperaturesand appearing a second transition (no so well de¢nedas in DMPC) at surfactin concentrations of 4.7mol% and higher.

The partial phase diagram obtained for this system(Fig. 7A) also displays a near-ideal behaviour for thesolidus line, with £uid phase immiscibility for surfac-tin concentrations of 1^2 mol% and above.

At di¡erence with DMPC, the vH of the maintransition of DMPG is greatly a¡ected by surfactin,decreasing from 6.9 kcal mol31 for pure DMPG toca. 3.5 kcal mol31 for a mixture containing 9.1 mol%of surfactin.

The e¡ect of Ca2� on the interaction of surfactinwith a negatively charged phospholipid such asDMPG was studied at pH 8.5, at which the L-Glu1

and L-Asp5 residues of the peptide are fully ionized[2]. The DSC results are shown in Fig. 8. It is ob-served that at 7 mM calcium concentration, additionof 2 mol% of surfactin results in a qualitativelystronger perturbation of the bilayer than in the ab-sence of calcium. Despite the Tc and vH not beingchanged much more in the presence than in the ab-

Fig. 5. X-Ray di¡raction patterns obtained for dispersions of pure DMPC and mixtures containing 1 mol% or 2 mol% of surfactin atdi¡erent temperatures (indicated on each panel). The data correspond to one representative experiment.

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sence of calcium (Table 1), the shape of the transi-tion is considerably more altered as observed in theDSC scans, showing a broader and less cooperativetransition. Probably, a higher calcium concentrationwould be required in order to observe a strongere¡ect. However, under our experimental conditions,total Ca2� had to be kept below ca. 10 mM(Ca2� :DMPG6 1:7), since binding of calcium toDMPG at Ca2� :DMPGs 1:7 results in formationof non-lamellar structures with the transition temper-ature shifted to considerably higher values [18].

In order to test the e¡ect of the head group of anegatively charged phospholipid, mixtures of surfac-tin and DMPS were studied by DSC. Interestingly,the e¡ect of incorporation of increasing amounts ofsurfactin into DMPS bilayers resulted in a weakere¡ect than with DMPG (results not shown).

It has been described [19] that DMPG samplesshow only a single and broad re£ection at the small

angle region, which indicates that the lamellar orderis poor. X-Ray di¡raction experiments of pureDMPG and DMPG containing 2 mol% and 5 mol%surfactin were performed, at temperatures below andabove the gel to liquid^crystalline phase transition(not shown). Our results con¢rmed those describedby Epand et al. [19], and also showed a single andbroad re£ection which did not allow obtaining anyfurther information about the bilayer properties andits modulation by surfactin.

3.3. Interaction of surfactin withdielaidoylphosphatidylethanolamine: e¡ect onlipid polymorphism

Fig. 9 shows the DSC pro¢les of mixtures of sur-factin with DEPE. Aqueous dispersions of DEPEcan undergo a gel to liquid^crystalline phase transi-

Fig. 6. DSC heating-scan thermograms for mixtures of surfactinwith DMPG. Experiments were carried out at pH 7.4. Eachsample contained 3 Wmol phospholipid and the appropriateamount of surfactin (mol% of total), which is expressed on thecurves.

Fig. 7. (A) Partial phase diagram for mixtures of surfactin withDMPG at pH 7.4. Filled symbols correspond to the solidus lineand open symbols to the £uidus line. G corresponds to a lamel-lar gel phase, and F and FP to lamellar £uid phases (see textfor explanation). Dashed lines separate di¡erent regions on thediagram. (B) Dependence of the enthalpy change of the gel toliquid^crystalline phase transition of mixtures of surfactin withDMPG at pH 7.4 on surfactin concentration. Data points cor-respond to the mean þ S.E. of three di¡erent experiments. Errorbars are shown when bigger than the symbols.

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tion in the lamellar phase and, in addition, a lamellarliquid^crystalline to hexagonal-HII transition [20].The gel to liquid^crystalline transition takes placearound 37³C and the lamellar to hexagonal-HII tran-sition at around 65³C, in agreement with previousdata [20]. The smaller vH of the transition to HII

phase is due to the £uid character of both the lamel-lar liquid^crystalline and the HII phases [21]. Incor-poration of increasing concentrations of surfactininto DEPE slightly shifts the Tc of the gel toliquid^crystalline phase transition to lower values.The transition to HII phase is also a¡ected, its Th

decreasing up to about 1 mol% surfactin and thenremaining constant.

Fig. 9 (inset) shows DSC scans corresponding tothe HII transition region, carried out at a higher sen-sitivity. It can be observed that the transition is stilldetectable by DSC at 6.5 mol% of surfactin. Increas-ing the concentration of the lipopeptide progressivelybroadens the transition, mainly toward the high tem-perature side.

The partial phase diagram for the system DEPE/surfactin (Fig. 10A) reveals a near-ideal behaviourfor the solidus line, whereas the £uidus line indicatesa £uid-phase immiscibility in the lamellar state (theline stays horizontal). The £uid lamellar/£uidlamellar+HII phase boundary reveals immiscibilityfrom 1 mol% surfactin and above, while the linecorresponding to £uid lamellar+HII/ HII boundaryprogressively shifts to higher values.

In order to characterize the phases present at eachparticular temperature in the absence and presence ofsurfactin, X-ray di¡raction was performed. The re-sults shown in Fig. 11 indicate that at 20³C DEPEadopts a lamellar structure and the incorporation ofup to 8 mol% of surfactin does not change this pat-tern. Furthermore, the d-spacing (67.2 Aî for pureDEPE) is not changed by e¡ect of surfactin. At50³C, DEPE adopts a lamellar £uid phase, as re-vealed by the marked decrease in d-spacing (54.6Aî ). Again, surfactin does not a¡ect either the typeof structure or the bilayer thickness at this temper-ature. Finally it can be observed that at 70³C, pureDEPE adopts a hexagonal-HII structure (lattice pa-rameter ratio 1:1/k3:1/k4:T) and the samples contain-ing up to 8 mol% of surfactin show essentially thesame di¡raction characteristics as pure DEPE.

4. Discussion

Interaction of surfactin with phosphatidylcholinemembranes will be ¢rst discussed. Di¡erential scan-

Fig. 8. DSC heating-scan thermograms for mixtures of surfactinwith DMPG in the absence (A) and presence (B) of 2 mol%surfactin with di¡erent calcium concentrations (numbers on thecurves, mM Ca2�). Experiments were carried out at pH 8.5.Each sample contained 3 Wmol phospholipid.

Table 1E¡ect of surfactin on the phase transition parameters of DMPG in the absence and presence of calcium at pH 8.5

3Ca2� +7 mM Ca2�

DMPG DMPG+2 mol% surfactin DMPG DMPG+2 mol% surfactin

Tc (³C) 23.8 þ 0.6 22.6 þ 0.3 23.4 þ 0.5 21.4 þ 0.3vH (kcal mol31) 6.9 þ 0.6 5.7 þ 0.4 4.9 þ 0.6 4.5 þ 0.4

The values represent the mean þ S.E. of three di¡erent measurements.

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ning calorimetry indicates that addition of surfactinto di¡erent phosphatidylcholines induces a progres-sive broadening of the transition peak and a shift ofthe Tc to lower values. These e¡ects are stronger inC14 and C16 phosphatidylcholines than in longerones, and particularly in DMPC, which is in agree-

ment with previous experiments using monolayertechniques [14], and are due to di¡erences in thespeci¢c interactions of surfactin with the di¡erentphosphatidylcholines under study, since the amountof lipopeptide associated with the lipid bilayer is thesame in all the three species (see Section 2). Ourresults indicate the establishment of molecular inter-actions between the phospholipid acyl chains and thesurfactin moiety, perturbing the cooperative behav-iour of the phospholipid, and can be explained bythe insertion of the surfactin molecules between thephosphatidylcholine molecules in the gel state. Theseresults are compatible with the surfactin moleculealigning itself with the prevailing direction of thephospholipid acyl chains, with the polar amino acid

Fig. 10. (A) Partial phase diagram for mixtures of surfactinwith DEPE. Filled circles correspond to the lamellar solidusline and open circles to the lamellar £uidus line. Filled squaresdepict the £uid lamellar/hexagonal-HII boundary and opensquares the £uid lamellar+hexagonal-HII/hexagonal-HII boun-dary. G corresponds to a lamellar gel phase, L is a lamellar £u-id phase and HII a hexagonal-HII phase (see text for explana-tion). (B) Dependence of the enthalpy change of the gel toliquid^crystalline phase transition (¢lled circles) and the lamellarto hexagonal-HII phase transition (open circles) of mixtures ofsurfactin with DEPE on surfactin concentration. Data pointscorrespond to the mean þ S.E. of three di¡erent experiments.Error bars are shown when bigger than the symbols.

Fig. 9. DSC heating-scan thermograms for mixtures of surfactinwith DEPE. Insert shows scans corresponding to the region ofthe hexagonal-HII transition carried out at a higher sensitivity.Each sample contained 3 Wmol phospholipid and the appropri-ate amount of surfactin (mol% of total), which is expressed onthe curves.

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ring placed near the lipid^water interface, where itcould establish interactions with water and the phos-pholipid polar head groups. According to this typeof interaction, the surfactin molecule will be kept atthe upper part of the phospholipid palisade, whichwould explain the small e¡ect observed on vH (Fig.4). Taken together, these results suggest that the sur-factin molecule is more active at the level of themembrane surface than at the level of the hydrocar-bon chains, which is in agreement with the importantinterfacial properties of surfactin [2].

Focusing on the mixtures of surfactin withDMPC, the appearance of a second peak in the ther-mograms, at concentrations of surfactin of 1 mol%and above (Fig. 2A) is attributed to a lateral phaseseparation of a surfactin-rich domain, in agreementwith the £uid-phase immiscibility observed in thecorresponding phase diagram (Fig. 3A). This typeof immiscibility has been reported before for mix-tures of phosphatidylcholines with free fatty acids[15], diacylglycerols [22] and retinoids [23], and canbe the result of strong interactions between surfactinmolecules, so that clusters are formed. Our resultscan also be explained by formation of surfactin-rich

domains which are laterally separated within theplane of the bilayer. Thus, in the region denotedG+F in the corresponding phase diagram (Fig.3A), G and F represent coexistence of gel and £uidstates, respectively, of, at least, a DMPC-rich phaseand a surfactin-rich phase, which is immiscible in a£uid bilayer. Coexistence of more phases cannot beruled out from our calorimetric data.

X-Ray di¡raction data clearly show that additionof surfactin at concentrations as low as 1^2 mol%induces a considerable increase of d-spacing inDMPC bilayers (Fig. 5), both in the gel and £uidstate, which might be caused by changes in eitherbilayer thickness or the water layer between adjacentlayers, or both. An increase in bilayer thicknesscould be the consequence of the intercalation of sur-factin-rich domains between the DMPC moleculeswithin the bilayer. The above-mentioned notionthat the surfactin molecule must reside near the bi-layer surface also supports this idea, since it wouldincrease the repulsive force between the surfaces ofadjacent bilayers resulting in an increase of d-spac-ing. A similar behaviour has been found before forthe antibiotic polypeptide alamethicin [24].

Fig. 11. X-Ray di¡raction patterns obtained for dispersions of pure DEPE and mixtures containing 3 mol% or 8 mol% of surfactin atdi¡erent temperatures (indicated on each panel). The data correspond to one representative experiment.

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Previous experiments using monolayer techniques[14] have failed to demonstrate the formation ofcomplexes of surfactin with phospholipids. Here wehave shown that, particularly in the case of the opti-mal chain length DMPC, surfactin-rich domains areformed, which are laterally separated within theplane of the bilayer. The fact that the £uidus linein the corresponding partial phase diagram (Fig.3A) remains horizontal from surfactin concentrationsof 1 mol% and above should be su¤cient to indicatethat these clusters possess a de¢ned stoichiometry.Nevertheless, more evidence should be obtained tounambiguously demonstrate formation of surfactin^phospholipid complexes and to determine its precisestoichiometry. However, the results shown here, i.e.,formation of surfactin clusters within the phospho-lipid bilayer, could also help to explain the ¢ndingthat surfactin forms ion-conducting pores in mem-branes [25].

Maget-Dana and Ptak [14] found formation ofdimers in the presence of calcium. Here, we havereported £uid-phase immiscibility in the absence ofcalcium, where dimers of surfactin with calcium can-not exist. The di¡erence might arise from the di¡er-ent approaches used. Whereas they used the mono-layer technique, we are dealing with phospholipidbilayers. Furthermore, whereas they mostly addedsurfactin to preformed monolayers, our samples con-tained the lipopeptide preincorporated with the phos-pholipid, prior to vesicle formation.

Surfactin, containing a L-glutamic and a L-asparticresidues, has an evaluated pK value at the air^waterinterface of 5.5^6.0 [2]. Since the actual interfacialpH is well known to be lower than the bulk pH[26], at pH 5.5 surfactin should be almost fully pro-tonated, whereas at pH 7.4 and 8.5 dissociation willincrease. We have found virtually the same resultsfor the interaction of surfactin with di¡erent phos-phatidylcholine species at pH 5.5, 7.4 and 8.5, prob-ably as expected given the uncharged nature of phos-phatidylcholine. However, in the case of a negativelycharged membrane, we expect the ionization state ofthe surfactin molecule to be rather relevant and thiswas checked in mixtures with DMPG. At pH 7.4, thee¡ect of neutral surfactin was tested and the patternwas qualitatively similar to that described above forDMPC. A marked di¡erence is that now, vH isstrongly decreased by the addition of surfactin (Fig.

7B), which is explained by a deeper insertion of thesurfactin molecule into the DMPG bilayer given thesmaller size of the polar head group of phosphatidyl-glycerol vs. phosphatidylcholine, in which higherhead to head repulsions should exist.

The presence of Ca2� at pH 7.4 did not change thepattern observed (results not shown), also supportingthe notion that at this pH the surfactin molecule isessentially protonated. In turn, at pH 8.5, addition ofCa2� resulted in a stronger e¡ect of surfactin onDMPG bilayer transition properties, with a muchmore broadened and less cooperative transitionthan in its absence (Fig. 8). Ionized surfactin bindsCa2� ions forming a surfactin^Ca2� 1:1 complex[27^29], which is expected to be located more deeplyinto the bilayer, resulting in a stronger interactionwith the phospholipid acyl chains. We have founda weaker e¡ect of calcium than that described beforefor the interaction of surfactin and phosphatidylser-ine in monolayers [14], and various explanations canbe given to the apparent discrepancies. First, we areusing here a di¡erent negatively charged phospho-lipid (namely phosphatidylglycerol vs. phosphatidyl-serine) and second, the calcium concentration wasmuch lower (7 mM vs. 20 mM); however, highercalcium concentrations could not be used in our sys-tem by the reasons stated above (see Section 3). Fi-nally, surfactin concentrations up to 20% were usedby Maget-Dana and Ptak [14], whereas the highestwe have studied was 9%. This might result in a stron-ger e¡ect of calcium in their case. Furthermore, inaddition, they used the monolayer technique whereaswe worked with phospholipid vesicles. To check therole of the negatively charged phospholipid used, westudied phospholipid vesicles composed of mixturesof surfactin with DMPS by means of DSC, and aneven weaker e¡ect than with DMPG was found,which gives more support to one of the two ¢rstpossibilities. Probably, small unilamellar vesicleswould be a good system to unambiguously evaluateCa2� e¡ects. However, the techniques we are dealingwith, namely X-ray di¡raction and di¡erential scan-ning calorimetry, are of no application with this typeof vesicles. We are considering other techniques so asto be able to follow surfactin e¡ects using unilamel-lar vesicles in general, and in SUV in particular.

The ability of hydrated lipids to adopt a variety ofphases in addition to the bilayer phase is a well-

BBAMEM 77594 3-5-99


documented fact. In this respect, the predilection ofunsaturated phosphatidylethanolamine for non-bi-layer con¢gurations has been recognized for sometime, representing one of the better known modelsfor studying lipid polymorphism [21]. We have chos-en DEPE as a convenient system for this purpose.

In the lamellar state, surfactin^DEPE mixturesshow £uid-phase immiscibility similar to that de-scribed above for mixtures with DMPC, consistentwith the same explanation given for this lipid. vH isslightly more a¡ected, up to 4 mol% surfactin, thanin the case of DMPC, indicating a deeper localiza-tion of the lipopeptide in DEPE membranes.

The lamellar to hexagonal-HII transition is broad-ened by e¡ect of surfactin, but only toward the hightemperature side. This indicates the existence of £uidlamellar and HII phases which are not miscible, asseen in the corresponding partial phase diagram (Fig.10A). The shifting of the complexion temperature ofthe lamellar to hexagonal-HII phase transition tohigher values also indicates that addition of surfactinto DEPE tends to destabilize the HII structure or, inother words, to stabilize the bilayer. This agrees wellwith the inverted cone molecular shape of the surfac-tin molecule, as opposed to the cone-shaped phos-phatidylethanolamine [21]. In fact, although surfactindoes not change the size of the hexagonal-HII tubulesas shown by X-ray di¡raction (Fig. 11), it does seemto induce a certain loss of organization of the HII

phase, since the re£ections lose sharpness and de¢ni-tion (Fig. 11).

In conclusion, in this work it has been shown thatsurfactin perturbs phospholipid bilayers in a di¡erentway, depending on lipid composition. The interac-tions seem to be optimal in the case of myristoylacyl chains, which have a similar length to the sur-factin hydrocarbon tail. The lipopeptide forms clus-ters with the phospholipids that are segregated orform domains within the bilayer. The ionized formof surfactin seems to be more deeply inserted into thebilayer when Ca2� is present, con¢rming the forma-tion of surfactin^Ca2� complexes, also in phospho-lipid bilayers. Finally, surfactin displays a bilayerstabilizing e¡ect in phosphatidylethanolamine sys-tems. Our results are compatible with the markedamphiphilic nature of surfactin and may contributeto explaining some of its interesting biological ac-

tions, for instance the formation of ion-conductingpores in membranes.

Acknowledgements

This work was ¢nancially supported by GrantPB96-1107 from DGES (Spain).

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