Henny Kell

Josef F Holzwarth

Christoph Boettcher

Richard K Heenan

Dr Joachim Vater
email: vater@chem.tu-berlin.de

Physicochemical studies of the interaction of the lipoheptapeptide surfactin with lipid bilayers of L-α-dimyristoyl phosphatidylcholine

Keywords: DMPC-vesicles, surfactin, differential scanning calorimetry, static light scattering, cryo-electron microscopy, small angle neutron scattering, "Poor Man's" temperature jump-relaxation spectroscopy

To understand the biological action of surfactin from Bacillus subtilis we investigated its effects on the phase transition of L-α-dimyristoyl phosphatidylcholine (DMPC)-vesicles from the crystalline to the fluid state using differential scanning calorimetry; light scattering; small angle neutron scattering and cryo-electron microscopy. DSC-thermograms revealed two phase transition peaks. Light scattering profiles showed two branches with characteristic hysteresis phenomena. With both techniques the same values of the phase transition temperatures T m1 and T m2 of 23.5 and 23 o C were obtained indicating two forms of DMPC-surfactin aggregates which could be visualized by cryo-electron microscopy. Until 4 mol% surfactin the vesicular form predominated, but was accompanied by bilayered membrane fragments by increasing the biosurfactant concentrations. At surfactin concentrations higher than 15 mol% smaller DMPC-surfactin micelles of ellipsoidal conformation were formed, as demonstrated by small angle neutron scattering. In addition, by "Poor Man's" -temperature jump-relaxation spectroscopy slow transients in the phase transition of vesicular DMPC-surfactin aggregates with relaxation times of 20-30 s were detected which presumably indicate the slow dissipation of intermediate lipid-and surfactin domains formed after the main phase transition on the way to the fluid state. This process is accelerated by surfactin.

Introduction

Microbial biosurfactants, as lipopeptides and glycolipids, show a high potential for medical and biotechnological applications. Surfactin, a lipopeptide which is produced by numerous Bacillus subtilis strains, is one of the most efficient biosurfactants so far known [START_REF] Arima | Surfactin, a crystalline peptide lipid surfactant produced by Bacillus subtilis: Isolation, characterization and its inhibition of clot formation[END_REF][START_REF] Vater | Lipopeptides, an attractive class of microbial surfactants[END_REF]. At a concentration of 20 μM (0.05%) it lowers the surface tension of water from 72 to 27 mN m -1 [START_REF] Arima | Surfactin, a crystalline peptide lipid surfactant produced by Bacillus subtilis: Isolation, characterization and its inhibition of clot formation[END_REF]. It is a cyclic lipoheptapeptide containing seven amino acids and a ß-hydroxy fatty acid of the following structure [START_REF] Kakinuma | Determination of amino acid sequence in surfactin, a crystalline peptidelipid surfactant produced by Bacillus subtilis[END_REF][START_REF] Kakinuma | Determination of fatty acid in surfactin and elucidation of the structure of surfactin[END_REF] Ring closure is achieved by lactone formation between the ß-hydroxyl group of the fatty acid component and the carboxyl group of the terminal L-Leu-residue [START_REF] Kakinuma | Determination of the location of lactone ring in surfactin[END_REF]. In solution the peptide ring of surfactin shows a "horse-saddle" topology [START_REF] Bonmatin | Solution three-dimensional structure of surfactin: A cyclic lipopeptide studied by 1 H-NMR, distance geometry, and molecular dynamics[END_REF]. Its two negatively charged amino acid residues L-Glu and L-Asp form a claw and function as binding sites for mono-and divalent cations, while its fatty acid side chain is extending at the opposite side of the ring. Natural

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surfactin is a complex mixture of closely related isoforms which vary in length and branching of its fatty acid component and/or the amino acid composition and sequence of its peptide ring [START_REF] Kowall | Separation and characterization of surfactin isoforms produced by Bacillus subtilis OKB 105[END_REF]. It contains ß-hydroxy fatty acids with side chains of 13-15 carbon atoms. Due to its amphiphilic structure it shows unique surface-, interface-and membrane-active properties [START_REF] Arima | Surfactin, a crystalline peptide lipid surfactant produced by Bacillus subtilis: Isolation, characterization and its inhibition of clot formation[END_REF][START_REF] Vater | Lipopeptides, an attractive class of microbial surfactants[END_REF][START_REF] Maget-Dana | Interfacial properties of surfactin[END_REF][START_REF] Maget-Dana | Interactions of surfactin with membrane models[END_REF][START_REF] Bernheimer | Nature and properties of a cytolytic agent produced by Bacillus subtilis[END_REF][START_REF] Razafindralambo | Foaming properties of surfactin, a lipopeptide biosurfactant from Bacillus subtilis[END_REF][START_REF] Deleu | Interfacial and emulsifying properties of lipopeptides from Bacillus subtilis, Colloids and Surfaces[END_REF] and a strong tendency for self-aggregation forming micelles and vesicles [START_REF] Ishigami | Significance of ß-sheet formation for micellization and surface adsorption of surfactin[END_REF][START_REF] Knoblich | Electron cryo-microscopic studies on micellar shape and size of surfactin, an anionic lipopeptide[END_REF].

Surfactin is distinguished by a broad spectrum of interesting biological activities which mainly depend on its membrane-active properties. For example, it shows potent antimicrobial [START_REF] Vater | Lipopeptides, an attractive class of microbial surfactants[END_REF][START_REF] Bernheimer | Nature and properties of a cytolytic agent produced by Bacillus subtilis[END_REF][START_REF] Vollenbroich | Antimycoplasma properties and application in cell culture of surfactin, a lipopeptide antibiotic from Bacillus subtilis[END_REF], antiviral [START_REF] Naruse | Pumilacidin, a complex of new antiviral antibiotics-production, isolation, chemical properties and biological activity[END_REF][START_REF] Itokawa | Structural and conformational studies of [Ile 7 ] and [Leu 7 ] surfactins from Bacillus subtilis natto[END_REF][START_REF] Vollenbroich | Mechanism of inactivation of enveloped viruses by the biosurfactant surfactin from Bacillus subtilis[END_REF][START_REF] Kracht | Antiviral and hemolytic activities of surfactin isoforms and their methylester derivatives[END_REF], antitumor [START_REF] Kameda | Antitumor activity of Bacillus natto V. Isolation and characterization of surfactin in culture medium of Bacillus natto KMD 2311[END_REF], and hypocholesterolemic [START_REF] Imai | Hypocholesterolemic effect of surfactin, a novel bacterial peptidelipid[END_REF] activities which are of high relevance in health care and biotechnology. As a membrane-active agent it lyses bacterial spheroplasts and protoplasts [START_REF] Bernheimer | Nature and properties of a cytolytic agent produced by Bacillus subtilis[END_REF] as well as mycoplasma and viral membrane envelopes [START_REF] Vollenbroich | Antimycoplasma properties and application in cell culture of surfactin, a lipopeptide antibiotic from Bacillus subtilis[END_REF][START_REF] Vollenbroich | Mechanism of inactivation of enveloped viruses by the biosurfactant surfactin from Bacillus subtilis[END_REF]. It is hemolytic by lysis of erythrocytes inhibiting fibrin clot formation [START_REF] Bernheimer | Nature and properties of a cytolytic agent produced by Bacillus subtilis[END_REF][START_REF] Kracht | Antiviral and hemolytic activities of surfactin isoforms and their methylester derivatives[END_REF]. Surfactin efficiently binds mono-and divalent cations [START_REF] Thimon | Interactions of surfactin, a biosurfactant from Bacillus subtilis, with inorganic cations[END_REF] and forms ion conducting pores in lipid bilayer membranes functioning as an ionophor for ion transport across biomembranes [START_REF] Sheppard | Ionic channels induced by surfactin in planar bilayer membranes[END_REF][START_REF] Thimon | Ionophorous and sequestering properties of surfactin, a biosurfactant from Bacillus subtilis[END_REF].

To understand the mechanisms of its specific biological actions detailed knowledge on its structure and physico-chemical properties is needed. It seems obvious that its amphiphilic nature and its specific surface-and membrane-active character play an important role in the expression of its biological activities. Particularly relevant are studies on the interaction of surfactin with artificial model membranes which have been performed by several authors [START_REF] Maget-Dana | Interactions of surfactin with membrane models[END_REF][START_REF] Grau | A study on the interaction of surfactin with phospholipid vesicles[END_REF][START_REF] Deleu | Nanometer scale organization of mixed surfactin/phophatidylcholine monolayers[END_REF][START_REF] Deleu | Imaging mixed lipid monolayers by dynamic atomic force microscopy[END_REF][START_REF] Heerklotz | Detergent-like action of the antibiotic peptide surfactin on lipid membranes[END_REF][START_REF] Carrillo | Molecular mechanism of membrane permeabilization by the peptide antibiotic surfactin[END_REF]. In previous work it has been demonstrated by electron microscopic analysis that surfactin inactivates mycoplasma [START_REF] Vollenbroich | Antimycoplasma properties and application in cell culture of surfactin, a lipopeptide antibiotic from Bacillus subtilis[END_REF] and enveloped viruses [START_REF] Vollenbroich | Mechanism of inactivation of enveloped viruses by the biosurfactant surfactin from Bacillus subtilis[END_REF][START_REF] Kracht | Antiviral and hemolytic activities of surfactin isoforms and their methylester derivatives[END_REF] by purely physicochemical mechanisms. The action of surfactin ultimately leads to complete desintegration of the envelope membranes. Therefore, the physicochemical approach to investigate the effects of surfactin on biomembranes seems most promising. Highly informative to explore the interaction of (bio)membranes with membrane-active compounds, In addition, of high importance is the investigation of the dynamic processes involved in the main phase transition of bilayer membranes and their interaction with membrane-active agents which need the application of fast relaxation techniques. By iodine-laser temperature jump studies in combination with time-resolved cryo-electron microscopic studies Groll et al.

[30] discriminated at least five fast processes being involved in the phase transition of lipid bilayer membranes which were observed in a time range of nanoseconds to seconds. In our present study we used the "Poor Man's" temperature jump technique to investigate slow transients in the time scale of 10-100 seconds which indicate the conversion of intermediate states formed, when the main phase transitions approach the fluid state.

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Materials and Methods

Materials

Surfactin was isolated from Bacillus subtilis OKB 105, as published by Baumgart et al. [START_REF] Baumgart | Identification of amino acid substitutions in the lipopeptide surfactin using 2D NMR spectroscopy[END_REF].

L-α-dimyristoyl phosphatidylcholine (DMPC) and L-α-dipalmitoyl phosphatidylcholine (DPPC) were obtained from Sigma-Aldrich (Taufkirchen, Germany).

Preparation of DMPC-vesicles:

DMPC was dissolved with ethanol in a concentration of 36 mM. The solution was slowly injected with a Hamilton syringe into 10 ml 2.5 mM Tricine buffer, 10 mM NaCl; pH 7.5 at a temperature of 28 o C. The solution of vesicles was dialyzed against the same Tricine buffer for 24 h at temperatures above the phase transition of the lipids. The vesicle preparation was stable for several days when stored above this temperature.

Methods

c) Cryo-electron microscopy of DMPC-surfactin aggregates

Electron microscopical investigations were carried out at a DMPC-concentration of 2.5 mM and surfactin concentrations varying between 0.05 -0. UK. Measurements were performed at 29 o C using the "white beam-time of flight" -method.

Incident neutron wavelengths in the range of 2 -10 Å were used at a frequency of 50 Hz [START_REF] Penfold | Shear-induced structures in concentrated surfactant micellar phases[END_REF].

The distance between sample and detector was 5 m. The scattering directions Q varied between 0.02 and 0. In this way the kinetics of processes in the phase transition which proceeded slower than 10 s could be monitored. The relaxation curves which followed a first-order reaction were recorded using a relaxation detection programme Hi-Tech-Scientific (Hi-Tech, Ltd., Salisburg. UK). Relaxation times τ ( s ) and light scattering amplitudes A (a.u.) were obtained by fitting relaxation curves with the evaluation programme Origin.

Results

To understand the mechanism of action of the lipoheptapeptide surfactin on biomembranes physicochemical studies of its interaction with DMPC-vesicles were performed which were taken as a model system for a bilayered phospholipid membrane. Surfactin was produced by the recombinant high producer strain B. subtilis OKB 105, as reported by Baumgart et al. [START_REF] Baumgart | Identification of amino acid substitutions in the lipopeptide surfactin using 2D NMR spectroscopy[END_REF]. Natural surfactin is a complex mixture of isoforms which show variations both in the length and branching of their fatty acid side chains as well as the amino acid composition and sequence of their peptide moiety. Its isoform composition has been investigated in detail by Kowall et al. [START_REF] Kowall | Separation and characterization of surfactin isoforms produced by Bacillus subtilis OKB 105[END_REF]. According to matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis it contained isoforms with 37% C13-, 39% C14-and 24% C15 ßhydroxy fatty acid components.

1.) Differential scanning calorimetry (DSC) measurements

The interaction of DMPC-vesicles with surfactin was investigated by differential scanning calorimetry (DSC). In Fig. As is apparent from Table 1, the phase transition temperatures T m1 and T m2 for both aggregation forms diminished with increasing surfactin concentration.

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This effect was more pronounced for T m1 , while T m2 decreased only slightly in the range of surfactin concentrations between 4 and 20 mol%.

The two peaks in the DSC-curve observed for DMPC-surfactin aggregates were fitted by two Gauss-curves from which the transition enthalpies ΔH cal1 and ΔH cal2 for the phase transition of both forms of DMPC-surfactin aggregates were determined as the areas under these peaks.

For peak 1 a small increase of ΔH cal1 from 18.7 kJ M -1 for pure DMPC-vesicles until approx.

20.6 kJ M -1 at a surfactin concentration of 4 mol% was observed. At higher lipopeptide concentrations ΔH cal1 decreased continuously to values of 1-2 kJ M -1 . In contrast ΔH cal2 attributed to phase transition 2 (peak 2) was nearly constant in the range of 5-20 mol% surfactin. In this range the scattering intensity detected in the down scan was slightly lower than the

2.) Investigation of phase transition processes in

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value obtained in the up scan. Also with increasing surfactin concentrations up to 3 mol% the level of light scattering intensity of the DMPC-surfactin-aggregates increased (Fig. 3A) in the down scan experiment. T m shifted to lower temperatures (Table 3) and the hysteresis effect was getting more and more pronounced (Fig. 3A). At 4 mol% surfactin an additional second scattering process was observed at temperatures above phase transition 1 indicating a second form of DMPC-surfactin interaction. This result is consistent with the appearance of a second peak in the DSC diagram at this surfactin concentration (phase transition 2). In contrast to the phase transition at T m1 in the down scan the light scattering intensity in the region of the phase transition for the second process decreased, when temperature was lowered. Also this phase transition was reversible, but a stronger hysteresis effect was observed than for process 1. The appearance of two hysteresis effects in the light scattering profile yields further evidence for two forms of DMPC-surfactin-aggregation. With increasing surfactin concentration until 10 mol% both hysteresis effects diminished and disappeared completely at approx. 15 mol% (Fig. 3C). Above this concentration the level of light scattering intensity at the starting temperature of the down scan was different from that reached at the end of the following up scan (Fig. 3D).

In Table 2

3.) Cryo-electron microscopic characterization of DMPC-surfactin-aggregates

Cryo-electron microscopic measurements have been performed to visualize the DMPC-as More detailed inspection of the membrane revealed a tendency to form segments in the order of 50 to 100 nm The bilayered organisation of the membrane was clearly resolved and gave a thickness of the lipid bilayer in the order of 4-5 nm which has been determined from sectional profiles according to the method given e.g. by Wang et al. [START_REF] Wang | Using cryo-EM to measure the dipole potential of a lipid membrane[END_REF].

In the presence of surfactin the overall structure of the vesicles was not significantly affected.

However, at surfactin concentrations higher than 4 mol% the vesicles were acommpanied by small entities in the order of 50 -100 nm, the number of which increased with increasing concentration of the biosurfactant. In most cases these particles display a high contrast bilayer profile identical to that measured for vesicles (Fig. 4C). In almost all cases areas of lower contrast are adjacent to the high contrast bilayer profile, which is a hint for the sheet-like nature of these entities.

Apparently, two kinds of structures, namely unilamellar vesicles (Fig. 4A-C) and sheet-like membrane fragments (Fig. 4C andD), could be visualized by cryo-electron microscopy representing the two aggregation forms detected by DSC-and light scattering measurements.

At surfactin concentrations above 15 mol% the DMPC-surfactin aggregates have not been resolved by cryo-electron microscopy probably due to size limitations. Therefore, for their structural characterization small angle neutron scattering studies were performed.

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4.) Small angle neutron scattering experiments

A third form of DMPC-surfactin aggregation was found at higher surfactin concentrations between 15-30 mol% of the lipopeptide. In this concentration range the DMPC-surfactin interaction was partially irreversible. This aggregation form was investigated by small angle neutron scattering. In Fig. 5 the scattering intensity I (Q) was measured as a function of the magnitude of the scattering vector Q. The scattering profile was fitted by theoretical model conformations. The best fitting of the experimental data was obtained for two ellipsoidal particles, one of an oblate form of dimensions R 1 = 5.1 and R 3 = 1.6 nm and a second one of a prolate conformation with R 1 = 2.9 and R 3 = 7 nm. The structure of DMPC-surfactin micelles has to be clarified in more detail by application of other physicochemical methods.

5.) Poor Man's Temperature (PT)-Jump measurements

The kinetics of phase transition processes in lipid bilayer membranes can be studied by methods developed for the time resolution of fast chemical reactions, as for example, by temperature-jump measurements. In this work we investigated specifically the slow transients in the main phase transition of DMPC-vesicles and DMPC-surfactin aggregates that were observed in a time scale of 10-100 s applying the PT-jump technique. Measurements were performed at a DMPC-concentration of 2.5 mM in the presence of 0 -4 mol% of surfactin.

Transients in the phase transition were detected by light scattering measurements at a wavelength of 350 nm. Then the sample cuvette equilibrated to the starting temperature was transferred manually to the thermostatted cuvette holder of the our Iodine-Laser-T-Jump equipment [START_REF] Groll | Temperature dependent intermediate structures during the main phase transition of dimyristoylphosphatidylcholine vesicles, a combined iodine laser temperature jump and time resolved cryoelectron microscopic study[END_REF]. Both down-and up-jumps were performed. The relaxation diagrams, i. e. the change in the scattering intensity ΔI as a function of time during the T-jump, were biphasic with a dominating fast part comprising about 90 % of ΔI and a minor slower part of approx.

10%. The kinetics of the fast phase could not be resolved, because the corresponding relaxation times in the range of micro-and milliseconds are by far lower than the time limit of the PT-instrument of approx. 5 s. However, the slow process could be resolved well. In the down-and up-jump relaxation times of 29.8 s were determined for pure DMPC-vesicles (see Fig. 6A).

PT-jump experiments performed in the presence of 1; 2 and 4 mol % surfactin are shown in Figs. 6B-D. As is apparent from Table 3, surfactin accelerates the slow process observed in the phase transition of DMPC-vesicles. At a lipopeptide concentration of 4 mol % τ was decreased to a value of 18.5 s. At higher surfactin concentrations, when the DMPC-vesicles were completely converted into sheet-like lamellar membrane fragments, the slow phase in the relaxation diagram disappeared.

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Discussion

Surfactin, a lipopeptide biosurfactant from Bacillus subtilis, diplays a characteristic horsesaddle topology [START_REF] Bonmatin | Solution three-dimensional structure of surfactin: A cyclic lipopeptide studied by 1 H-NMR, distance geometry, and molecular dynamics[END_REF][START_REF] Tsan | Structure and dynamics of surfactin studied by NMR in micellar media[END_REF]. It is an attractive natural compound showing a broad spectrum of antimicrobial [START_REF] Vater | Lipopeptides, an attractive class of microbial surfactants[END_REF][START_REF] Bernheimer | Nature and properties of a cytolytic agent produced by Bacillus subtilis[END_REF][START_REF] Vollenbroich | Antimycoplasma properties and application in cell culture of surfactin, a lipopeptide antibiotic from Bacillus subtilis[END_REF] and antiviral [START_REF] Naruse | Pumilacidin, a complex of new antiviral antibiotics-production, isolation, chemical properties and biological activity[END_REF][START_REF] Itokawa | Structural and conformational studies of [Ile 7 ] and [Leu 7 ] surfactins from Bacillus subtilis natto[END_REF][START_REF] Vollenbroich | Mechanism of inactivation of enveloped viruses by the biosurfactant surfactin from Bacillus subtilis[END_REF][START_REF] Kracht | Antiviral and hemolytic activities of surfactin isoforms and their methylester derivatives[END_REF] activities. Therefore, it is of great medical and biotechnological interest. The action of surfactin on microorganisms and viruses mainly depends on its membrane-active properties [START_REF] Vollenbroich | Antimycoplasma properties and application in cell culture of surfactin, a lipopeptide antibiotic from Bacillus subtilis[END_REF][START_REF] Vollenbroich | Mechanism of inactivation of enveloped viruses by the biosurfactant surfactin from Bacillus subtilis[END_REF]. Because of its amphiphilic structure it is able to efficiently penetrate into biomembranes, ultimately leading to their disintegration [START_REF] Vollenbroich | Antimycoplasma properties and application in cell culture of surfactin, a lipopeptide antibiotic from Bacillus subtilis[END_REF][START_REF] Vollenbroich | Mechanism of inactivation of enveloped viruses by the biosurfactant surfactin from Bacillus subtilis[END_REF]. Therefore, studies on the surfactin-biomembrane interaction are an indispensible Cryo-electron microscopic and small angle neutron scattering studies proved the existence of the three aggregation forms indicated by DSC and light scattering experiments. Particularly cryo-electron micrographs illustrate aggregation forms 1 and 2 (Fig. 4A-D). Pure DMPC-gave vesicles with a clearly resolved bilayer profile. Additionally, a segmentation of the membrane in the order of 50 -100 nm can be seen. This effect is probably due to different packing density between the outer and inner membrane layer which has been proven in small unilamellar vesicles of DMPC by NMR [START_REF] Mclaughlin | Temperature and frequency dependence of[END_REF] and was later refered to be due to electrostatic effects [37]. At surfactin concentrations higher than 4 mol% the number of the vesicular particles decreased. They were progressively accompanied by smaller objects with a length of 50-100 nm, which partially provided a bilayer profile similar to that observed in the vesicles.

Although at first sight the high contrast of this bilayer profile suggests the fragments to show a rod-like molecular architecture, a closer examination indicated that the structure of the fragments must be understood of being organized by more or less planar bilayered membrane sheets. Since the objects have been trapped in the layer of the solvent upon vitrification in different spatial orientation with respect to the incident electron beam, differences in shape and structure depending on the view angle were revealed. Arrows in Fig. 4C andD There is also a progressive decrease of the phase transition temperature and a broadening of the DSC-peaks with increasing surfactin concentration. In contrast to DMPC, however, the second DSC-peak arising at 5-10 mol% surfactin was found at lower temperatures than the peak for the vesicular form. Both the shifts of T m1 and ΔHcal 1 at increasing surfactin concentration were smaller than in the case of the DMPC-surfactin system. These features imply a weaker interaction of surfactin with DPPC than with DMPC which can mainly be explained by the different lengths of their fatty acid side chains. DMPC has a C14-side chain which matches the length of a major portion of the ß-hydroxy fatty acid components of natural surfactin. This seems more favorable in the interaction with the myristoyl-acyl chains of DMPC, while the molecular forces with the C16palmitoyl-side chains of DPPC are weaker. Similar results had previously been reported by Grau et al. [START_REF] Grau | A study on the interaction of surfactin with phospholipid vesicles[END_REF].

Also the light scattering profiles observed for the phase transitions of DMPC and DPPC were qualitatively similar showing two branches with characteristic hysteresis phenomena. For the vesicular form of the DPPC-surfactin aggregates observed until a surfactin concentration of approx. 5 mol% again the scattering intensity increased in the down-scan experiment, while the opposite behaviour was found above this concentration. As for DMPC irreversible lipidbiosurfactant interaction ocurred at surfactin concentration higher than 20 mol% which finally resulted in mixed micellar aggregates of low light scattering intensities.

Kinetic studies on the main phase transition of unilamellar lipid vesicles by iodine laser temperature jump experiments in combination with time resolved cryo-electron microscopy were performed by Groll et al. [START_REF] Groll | Temperature dependent intermediate structures during the main phase transition of dimyristoylphosphatidylcholine vesicles, a combined iodine laser temperature jump and time resolved cryoelectron microscopic study[END_REF] for DMPC vesicles. These authors developed a model for the hierarchie of the dynamic processes occuring during the phase transition from the crystalline to the fluid state, as illustrated in Fig. 7. A sequence of at least five events could be

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resolved. The initial fast relaxation processes observed in the nano-to microsecond range were attributed to kink formation of the hydrocarbon chains followed by head group rotation.

Rotational isomers of DMPC are formed by conversion of the all trans-into the cis -trans conformation increasing their lateral mobility. In the phase transition starting from the crystalline hexagonal array intermediate lipid clusters/ domains are formed which arise in the micro-to millisecond time scale followed by a slow conversion of the lipid molecules into the fluid state. This process has not yet been explored in detail. According to the model of Groll et el. [START_REF] Groll | Temperature dependent intermediate structures during the main phase transition of dimyristoylphosphatidylcholine vesicles, a combined iodine laser temperature jump and time resolved cryoelectron microscopic study[END_REF] the supramolecular intermediates appear as patches segregated within the bilayer.

They can be made visible by time-resolved cryo-electron microscopy. The detection and kinetic analysis of these unknown slow processes was another aim of this paper. For this purpose the "Poor Man's" temperature jump technique was used by which transients in the range of seconds and the influence of surfactin on these slow relaxation processes could efficiently be investigated. They were only observed in the case of pure DMPC-vesicles and vesicular DMPC-surfactin aggregates at low surfactin concentrations until 4 mol%. At higher lipopeptide concentrations, when the vesicles were disintegrated into lamellar membrane fragment sheets, the slow phase in the relaxation diagram completely disappeared.

Taking together the results of our thermodynamic, kinetic and structural studies the following features of the DMPC-surfactin interaction can be proposed: In all experiments the surfactin concentration in solution was higher than the critical micelle concentration CMC of approximately 10 μM [START_REF] Ishigami | Significance of ß-sheet formation for micellization and surface adsorption of surfactin[END_REF][START_REF] Heerklotz | Detergent-like action of the antibiotic peptide surfactin on lipid membranes[END_REF]. Therefore, it has to be assumed that always assemblies of the lipopeptide were taken up into the DMPC-lipid membrane. Our thermodynamic and kinetic data imply that at surfactin concentrations lower than 4 mol% lipopeptide molecules are dispersed within the bilayer membrane and interact with the adjacent lipid molecules.

Because of the bulky polar head part of surfactin the hexagonal array of the lipid bilayer is gradually destabilized increasing the fluidity of hydrocarbon side chains of the lipid This view is consistent with the model of Deleu et al. [START_REF] Deleu | Nanometer scale organization of mixed surfactin/phophatidylcholine monolayers[END_REF][START_REF] Deleu | Imaging mixed lipid monolayers by dynamic atomic force microscopy[END_REF] mainly derived from atomic force microscopy. Under these conditions ΔH cal1 strongly decreased and the slow phase of the relaxation profiles disappeared. ΔH cal2 characteristic for the phase transition of the lamellar DMPC-surfactin sheet-like lamellar fragments was nearly independent of the surfactin concentration indicating only weak interactions between lipid and surfactin molecules.

According to the data reported by Grau et al. [START_REF] Grau | A study on the interaction of surfactin with phospholipid vesicles[END_REF] the appearance of the second peak in the DSC-thermogram can be attributed to a lateral phase segregation of surfactin-rich domains in agreement with their fluid-phase immiscibility. Presumably surfactin integrates into the lamellar membrane fragments by growth of the biosurfactant clusters rather than by direct interaction with the lipid molecules. There is a coexistence between DMPC-rich and surfactin-rich domains, as demonstrated by Grau et al. [START_REF] Grau | A study on the interaction of surfactin with phospholipid vesicles[END_REF] and Deleu et al. [START_REF] Deleu | Nanometer scale organization of mixed surfactin/phophatidylcholine monolayers[END_REF][START_REF] Deleu | Imaging mixed lipid monolayers by dynamic atomic force microscopy[END_REF]. Finally, at surfactin concentrations higher than 15 mol% conversion of the membrane fragments into smaller DMPC-surfactin mixed micelles of ellipsoidal conformation occured which were characterized by small angle neutron scattering experiments. Consistent with the phenomena observed for the interaction of surfactin with phospholipid model membranes of DMPC and DPPC the biological action of surfactin on mycoplasma [START_REF] Vollenbroich | Antimycoplasma properties and application in cell culture of surfactin, a lipopeptide antibiotic from Bacillus subtilis[END_REF] and enveloped viruses [START_REF] Vollenbroich | Mechanism of inactivation of enveloped viruses by the biosurfactant surfactin from Bacillus subtilis[END_REF] is exerted by its micellar rather than by its monomolecular form. Below the critical micelle concentration only small inactivation effects were observed.

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Fig. 1

 1 Fig. 1 Structure of surfactin

  are studies of the influence of such agents on the phenomena involved in the temperature dependent phase transition from the crystalline into the fluid state. To clarify the specific action of surfactin we performed such studies with DMPC bilayer model membranes using thermodynamic, kinetic and structural methods. Differential scanning calorimetry (DSC) and light scattering provided evidence for two forms of DMPC-surfactin aggregates which could be visualized as vesicular particles and bilayered lamellar membrane fragments by cryo-electron microscopy, while small angle neutron scattering measurements revealed a third micellar form which presumably represents mixed DMPC-surfactin micelles.

a)

  Differential scanning calorimetry (DSC) DSC-experiments with DMPC vesicles were performed with a MicroCalMC-2 instrument (MicroCal, Northampton, MA, USA) at a lipid concentration of 2.5 mM in 2.5 mM Tricine buffer, 10 mM NaCl; pH 7.5 between 10 and 35 o C. The scan rate was 30 o C h -1 . Heat capacity c p (J o C -1 ) was measured as a function of temperature ( o C). Cp was converted into molar heat capacity c p m (kJ mol -1 o C -1 ) accounting for the cell volume (V = 1.225 ml) and the sample concentration. The two peaks in the DSC curves measured for DMPC were fitted by Gauss functions. The phase transition temperature T m ( o C) of the DMPC vesicles was obtained from the Gauss curves after baseline subtraction at the highest molar heat capacity c p m . The area under the DSC peaks corresponds to the molar enthalpy change ΔH cal (kJ mol -1 ) for the phase transition. A C C E P T E D M A N U S C R I P T b) Static light scattering Light scattering measurements on solutions of DMPC-vesicles were performed with a Shimadzu spectrofluorophotometer RF-5000 using a 150 W Xenon lamp as light source at a wavelength of 300 nm. The scattered light was observed at an angle of 90 o . DMPC in a concentration of 2.5 mM was dissolved in 2.5 mM Tricine buffer, 10 mM NaCl; pH 7.5. Surfactin was injected above the phase transition temperature T m in concentrations between 0.025 and 0.75 mM (1-30 mol%). To study the phase transition phenomena, the intensity of the scattered light was measured as a function of temperature starting with a "Down scan" in a temperature range of 40 to 15 o C followed by an "Up scan" over the same temperature interval. The heating rate was 12 o C h -1 . A Haake F3-C-thermostat was used for the temperature program. Temperature was controlled by thermo-couples of type Therm 2244-1, NTC Typ C, Ahborn Mess-und Regelungstechnik (Holzkirchen, Germany).

25 Å - 1 .

 251 Prior to the measurements a temperature treatment of the DMPCsurfactin solution in the range of 15 -40 o C in up-and down-mode was performed in order to obtain stable preparations. The measured scattering curves I (Q) were fitted by theoretical models of oblate and prolate ellipsoidal particles. e) Relaxation spectroscopy; "Poor Man's" Temperature Jump (PT-Jump) PT-Jump-measurements on DMPC-surfactin solutions were performed at a DMPCconcentration of 2.5 mM and surfactin concentrations of 0.025 -0.1 mM (1-4 mol%) in 2.5 A C C E P T E D M A N U S C R I P T mM Tricine buffer, 10 mM NaCl; pH 7.5. Samples were filled into 2 x 10 mm Quartz cuvettes and thermostatted for 5 min. For 1, 2 and 3 mol% surfactin the T-jump downwards was performed from 32 to 14 o C and correspondingly the up-jump from 14 to 32 0 C. For 4 mol% surfactin the down-jump was from 35 to 24 o C and the up-jump from 24 to 35 o C, respectively. PT-jump-experiments were started with a down-jump. For these measurements two thermostats were used. With the first one the initial temperature was adjusted, while the second one controlled the temperature in the thermostatable sample holder of our Iodine-Laser-T-Jump equipment [30] which was used in the time resolved light scattering mode at 350 nm during the T-jump. Relaxation signals were detected within a time interval of 100 s.

  2 A-D the effect of surfactin on the phase transition phenomena of DMPC vesicles is demonstrated. The DSC diagram of pure DMPC vesicles shows a single DSC-peak 1 with a maximum at the phase transition temperature T m1 = 23.5 o C (phase transition 1). In the presence of surfactin peak broadening and a shift of T m to lower temperatures was observed. At 4 mol% surfactin a shoulder appeared at the right flank of the DSC peak which at higher surfactin concentrations in the range of 10-15 mol% developed to a separate peak 2 (phase transition 2), while peak 1 was fading out. It completely disappeared at 20 mol% surfactin. At 4 mol% surfactin T m2 = 23 o C was extrapolated. The two peaks in the DSC diagram indicate phase transition processes that can be attributed to two forms of DMPC-surfactin aggregates.

  DMPC-surfactin vesicles by static light scatteringIn addition to DSC, the effect of surfactin on the phase transition of DMPC-vesicles was investigated by static light scattering. The scattering intensity I of the DMPC-vesicles around the phase transitions was monitored as a function of temperature in the range of 16-30 o C. The obtained results are shown in Fig.3A-D. Scanning was started at temperatures above the phase transition point. For pure DMPC-vesicles (Fig.3A) light scattering intensity increased with decreasing temperature (down scan). S-like scattering profiles were observed showing a strong increase of the intensity at the phase transition and a smaller increase at temperatures below and above the transition. The inflection point corresponds to the phase transition temperature T m1 . The phase transition process was reversible, i.e. in the following up scan experiment the initial level of light scattering intensity I was reached at the end temperature of 29 o C, but a small hysteresis effect was observed at temperatures around the phase transition.

  the data of the influence of surfactin on the transition temperatures T m1 and T m2 for both forms of DMPC-surfactin aggregates are summarized. In down scan experiments T m1 is starting at 23.4 o C in the case of pure DMPC vesicles decreasing to a value of 19.2 o C at 10 mol% surfactin, when the first hysteresis effect was disappearing. The second phase transition started at 4 mol% showing a transition temperature T m2 = 23.5 o C. These results are in excellent agreement with the DSC data.

  Fig. 4A-D were taken with a Philips Tecnai F20 transmission electron microscope, while maintaining a sample temperature of 89K . At temperatures higher than 25 o C pure DMPC

  prerequisite to understand its biological action. With this aim we investigated the effects of surfactin on DMPC vesicles as a model of a phospholipid bilayer membrane by physicochemical techniques. Differential scanning calorimetry (DSC) and static light scattering as well as methods for the structural characterization of lipid-surfactin aggregates, as cryo-electron microscopy and small angle neutron scattering (SANS) were applied. The results obtained by DSC-and light scattering experiments are in excellent agreement. Both techniques give evidence for two forms of DMPC-surfactin aggregates. DSCthermograms show two phase transition peaks, while light scattering profiles indicate two branches with pronounced hysteresis phenomena. Nearly the same values of the transition temperatures T m1 and T m2 of 23 -23.5 o C have been determined by both methods. With increasing surfactin concentrations DSC-measurements show a progressive broadening of the phase transition peaks and a shift of T m1 and T m2 to lower values indicating a decrease in the strength and cooperativity of the lipid-lipid and lipid-surfactin interaction in the bilayer membrane. The second peak in the thermogram and the second branch of the light scattering profiles appeared at surfactin concentrations above 4 mol%. T m2 showed a smaller decrease with increasing surfactin concentration than T m1 . Both aggregation forms revealed characteristic differences in the dependence of ΔH cal on the surfactin concentration. For ΔH cal1 A C C E P T E D M A N U S C R I P T an initial increase was observed until 4 mol% followed by a pronounced decrease until approximately 15 mol%, while ΔH cal2 for the phase transition of the second form remained nearly constant between 4 and 30 mol% surfactin. At surfactin concentrations higher than 20 mol% irreversible effects were observed. Under these conditions the original level of light scattering at the start of the down scan was not reached again at the end of the opposite upscan. At 30 mol% surfactin in the up-scan experiment no change in the scattering intensity occured, i. e. the DMPC-surfactin system was residing in the state reached after the down scan. These effects indicate a third form of DMPC-surfactin aggregates.

  clearly point to tilted views of such membrane fragments displaying the sheet-like topview A C C E P T E D M A N U S C R I P T characteristics with the bilayered side-view profile. In Fig. 4C, however, notably the topview projection image reveals some regular striations indicating additional undulations of the membrane fragments (encircled) typically observed for highly flexible membranes, which is also clearly visble in a more tilted object in Fig. 4C, indicated by the dashed arrow. Surfactin, therefore, is thought to act like a scissor shearing off smaller membrane areas from the vesicle surface, presumably along the vesicle segmentation "crestlines". Such flexible membrane fragments were observed in different orientation by cryo-TEM. At surfactin concentrations higher than 20 mol%, at which irreversible effects have been observed by DSC and light scattering measurements, small particles appeared which could not properly be resolved by cryo-electron microscopy. Therefore, their size and conformation were investigated by small angle neutron scattering. The scattering profiles were fitted well by two models which cannot yet be discriminated. These particles presumably represent mixed micelles of DMPC and surfactin of ellipsoidal form. This process was accompanied by a strong decrease in light scattering. Similar phenomena have also been reported for the solubilization of lipid bilayers by synthetic surfactants which are accompanied by morphological changes of the bilayer and the emergence of mixed micelles [38]. As is the case for the interaction of surfactin with DMPC lipid vesicles sometimes intermediates in form of disks or sheet-like bilayer fragments occurred. By incorporation of the biosurfactant into the DMPC-bilayer intermediate sheet-like lamellar fragments start to form that coexist with the vesicles, until they are completely disintegrated. At higher surfactin concentrations finally ellipsoidal mixed micelles are formed. Preliminary DSC and light scattering studies on the interaction of surfactin with L-αdipalmitoyl phosphaditylcholine (DPPC) have been performed. Similar phenomena have been observed as for DMPC vesicles, but the effects appeared at appreciably higher surfactin A C C E P T E D M A N U S C R I P T concentrations (data not shown). Also here the thermograms and the light scattering profiles indicate two forms of DPPC-surfactin aggregates.

  these conditions an increase of ΔH cal1 , the appearance of a slow phase in the relaxation diagrams of the PM-T-jump experiments and a decrease of the relaxation times with increasing surfactin were observed. This interpretation is in agreement with mono-and bilayer studies[START_REF] Maget-Dana | Interactions of surfactin with membrane models[END_REF][START_REF] Grau | A study on the interaction of surfactin with phospholipid vesicles[END_REF] which have shown that surfactin is miscible with phospholipids at low lipopeptide concentration. Increasing the surfactin concentration above 4 mol% patches of surfactin clusters/domains persist ultimately giving rise to a disruption of the DMPC vesicles into lamellar fragment sheets. In this state surfactin is almost immiscible within the leaflet.

LegendsFig. 1 Fig. 2 MolarFig. 3 LightFig. 4 Cryo

 1234 Fig.1 Structure of surfactin

Fig. 5 Small

 5 Fig. 5

Fig. 6 PoorFig. 7 Model

 67 Fig.6

Table 1 .

 1 The effect of surfactin on T m and ΔH cal of the endothermic phase transition of DMPC-vesicles determined by differential scanning calorimetry (DSC) ± 0.0 23.0 ± 0.1 20.6 ± 0.3 13.7 ± 0.2 7.1 ± 0.1 0.125 5 21.9 ± 0.2 23.0 ± 0.1 20.4 ± 0.7 10.9 ± 0.4 11.5 ± 0.4 0.150 6 21.4 ± 0.1 22.7 ± 0.1 19.4 ± 1.1 9.6 ± 0.6 11.8 ± 0.7 0.175 7 20.7 ± 0.2 22.5 ± 0.1 19.3 ± 1.1 7.8 ± 0.4 11.8 ± 0.7 0.200 8 20.1 ± 0.2 22.4 ± 0.1 18.4 ± 1.1 6.3 ± 0.4 11.8 ± 0.7 T m1 and T m2 , transition temperatures of the first and second DSC-peak; ΔH cal total , total transition enthalphy; ΔH cal1 and ΔH cal2 , transition enthalpies of the first and second DSC-peak.

	Surfactin concentration	T m1	T m2	ΔH cal total	ΔH cal1	ΔH cal2
	mM	mol%	°C	°C	kJ / M	kJ / M	kJ / M
	0.000	0	23.5 ± 0.0		18.7 ± 0.3 18.7 ± 0.3
	0.025	1	23.5 ± 0.1		18.7 ± 0.3 18.7 ± 0.3
	0.050	2	23.0 ± 0.1		19.7 ± 0.2 19.7 ± 0.2
	0.075	3	22.3 ± 0.1		20.1 ± 0.3 20.1 ± 0.3
	0.100 22.2 0.250 4 10 18.1 ± 0.2 22.0 ± 0.1 15.5 ± 0.9 3.8 ± 0.2 11.5 ± 0.7
	0.350	14	16.8 ± 0.2 21.5 ± 0.1 14.4 ± 0.8 1.8 ± 0.1 11.4 ± 0.7
	0.375	15		21.1 ± 0.1 12.6 ± 0.7 1.0 ± 0.1 11.4 ± 0.7
	0.500	20		21.0 ± 0.1 11.1 ± 0.7		11.1 ± 0.7
	0.750	30		21.0 ± 0.1 9.7 ± 0.6		9.7 ± 0.6