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Chemistry and Physics of Lipids 158 (2009) 46–53

Contents lists available at ScienceDirect

Chemistry and Physics of Lipids

journa l homepage: www.e lsev ier .com/ locate /chemphys l ip

nteractions of a bacterial biosurfactant trehalose lipid with phosphatidylserineembranes

ntonio Ortiza, José A. Teruela, María J. Espunyb, Ana Marquésb, Ángeles Manresab,rancisco J. Arandaa,∗

Departamento de Bioquímica y Biología Molecular-A, Facultad de Veterinaria, Universidad de Murcia, Campus de Espinardo, E-30100 Murcia, SpainLaboratorio de Microbiología, Facultad de Farmacia, Universidad de Barcelona, Joan XXIII s/n, E-08028 Barcelona, Spain

r t i c l e i n f o

rticle history:eceived 17 September 2008eceived in revised form 22 October 2008ccepted 4 November 2008vailable online 13 November 2008

eywords:iosurfactant

a b s t r a c t

Trehalose lipids are biosurfactants produced by rhodococci that, in addition to their well known potentialindustrial and environmental uses, are gaining interest in their use as therapeutic agents. The study of theinteraction of biosurfactants with membranes is important in order to understand the molecular mech-anism of their biological actions. In this work we look into the interactions of a bacterial trehalose lipidproduced by Rhodococcus sp. with dimyristoylphosphatidylserine membranes by using differential scan-ning calorimetry, X-ray diffraction and infrared spectroscopy. Differential scanning calorimetry and X-raydiffraction show that trehalose lipid broadens and shifts the phospholipid gel to liquid-crystalline phase

rehalose lipidhosphatidylserineSC-ray diffractionT-IR

transition to lower temperatures, does not modify the macroscopic bilayer organization and presentsgood miscibility both in the gel and the liquid-crystalline phases. Infrared experiments show that tre-halose lipid increases the fluidity of the phosphatidylserine acyl chains, changed the local environmentof the polar head group, and decreased the hydration of the interfacial region of the bilayer. Trehaloselipid was also able to affect the thermotropic transition of dimyristoylphosphatidyserine in the presence

suppuctur

of calcium. These resultsbilayers and produces str

. Introduction

Biosurfactants are a structurally diverse group of amphipathicurface-active molecules of microbial origin. Biosurfactants areaining an enormous interest because they show improved prop-rties as compared to their chemically synthesized counterparts;hey are less toxic, and more biodegradable and diverse (Rosenbergnd Ron, 1999; Lang, 2002). The biologically most important groupf glycolipids are the plant galactolipids, which have been showno strongly modify the physical behaviour of phospholipids mem-ranes (Popova and Hincha, 2005). Glycolipids of bacterial originre widespread biosurfactants which contain carbohydrates inombination with long-chain aliphatic acid or hydroxyl aliphaticcids. An important group of biosurfactant glycolipids includeshe rhamnolipids secreted by Pseudomonas aeruginosa, which haveeen shown to affect the structural properties of membrane

hospholipids (Ortiz et al., 2006; Sánchez et al., 2006; Arandat al., 2007a). Other important group of glycolipid biosurfac-ants is formed by trehalose-containing glycolipids (Asselineaund Asselineau, 1978). These trehalose lipids are mainly produced

∗ Corresponding author. Tel.: +34 968 364760 fax: +34 968 364147.E-mail address: fjam@um.es (F.J. Aranda).

009-3084/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.chemphyslip.2008.11.001

ort the idea that trehalose lipid incorporates into the phosphatidylserineal perturbations which might affect the function of the membrane.

© 2008 Elsevier Ireland Ltd. All rights reserved.

by rhodococci and present such interesting physicochemical andbiological properties that a number of industrial and environmen-tal applications have been proposed for them (Lang and Philp,1998).

Besides the environmental and industrial use of trehalose lipids,a significant potential application is emerging as therapeutic agents(Banat et al., 2000; Rodrigues et al., 2006). Trehalose lipids havebeen reported to have antiviral properties (Azuma et al., 1987;Hoq et al., 1997). It has been shown that trehalose lipids haveexcellent growth inhibition and differentiation-inducing activitiesagainst human leukaemia cells (Isoda et al., 1996, 1997b; Sudo et al.,2000). They also inhibit the activity of phospholipids- and calcium-dependent protein kinase C of HL60 cells (Isoda et al., 1997a) andshow immunomodulating activity (Kuyukina et al., 2007).

Regardless of these intriguing properties, many of which mightresult from the interaction of biosurfactants with membranes, veryfew works have been focused on the study of the molecular basisunderlying their membrane related biological actions. In order toget insight into the molecular interaction between these biosurfac-

tants and the lipidic component of biological membranes, we havestudied the effect of trehalose lipid on the most important mem-brane phospholipids. We have shown that trehalose lipid increasesthe fluidity of phosphatidylcholine membranes and forms domainsin the fluid state (Aranda et al., 2007b). Recently we have found

A. Ortiz et al. / Chemistry and Physic

ttgiApeptWlia(scftded

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2

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2

a(tpva

Fig. 1. Chemical structure of Rhodococcus sp. trehalose lipid.

hat trehalose lipid exhibits and important dehydrating effect onhe interfacial region of saturated phosphatidylethanolamines andreatly promotes the formation of the inverted hexagonal HII phasen unsaturated phoshpatidylethanolamines (Ortiz et al., 2008).lthough zwiterionic phospholipids, like phosphatidylcholine andhosphatidylethanolamine, are the major lipid components ofukaryotic membranes, anionic phospholipids are also consistentlyresent in substantial amounts and are known to be critical struc-ural and functional components of biomembranes (Buckland and

ilton, 2000). Phosphatidylserine is the major anionic phospho-ipid present in eukaryotic membranes. The negative surface chargemparted by phosphatifdylserine is required for the binding andctivation of various peripheral and integral membrane proteinsMcLaughlin and Aderem, 1995; Newton, 1995). In order to under-tand the influence of this biosurfactant on the phosphatidylserineomponent of membranes, we have purified a trehalose lipid (Fig. 1)rom Rhodococcus sp. and made a detailed study of the effect ofhe glycolipid on the thermotropic and structural properties ofimyristoylphosphatidylserine (DMPS) membranes, using differ-ntial scanning calorimetry (DSC), small and wide angle X-rayiffraction (SAX and WAX) and infrared spectroscopy (FT-IR).

. Materials and methods

.1. Materials

Dimyristoylphosphatidylserine, sodium salt (DMPS) was pur-hased from Avanti Polar Lipids Inc. (Birmingham, AL). All thether reagents were of the highest purity available. Purified wateras deionised in a Milli-Q equipment from Millipore (Bedford,A), and filtered through 0.24 �m filters prior to use. Stock

olutions of DMPS and the trehalose lipid were prepared inhloroform/methanol (8:1) and stored at −20 ◦C. Phospholipid con-entrations were determined by phosphorous analysis (Böttcher etl., 1961).

.2. Trehalose lipid production and purification

Strain 51T7 was isolated from an oil-contaminated soil sam-le after culture enrichment with kerosene, and was identified ashodococcus sp. (Espuny et al., 1995). This strain was maintainedy fortnightly cultures on Trypticase Soy Agar (Pronadisa, Spain)nd preserved in cryovials at −20 ◦C. Biosurfactants were produced,urified and it structure was characterized as described beforeEspuny et al., 1995, 1996).

.3. Differential scanning calorimetry

Samples for DSC were prepared by mixing the appropriatemounts of DMPS and trehalose lipid in chloroform/methanol

8:1). In the case of measurements in the presence of calciumhe appropriate amount of ionophore A23187 in ethanol to give ahospholipid/ionophore ratio of 500:1, was also added. The sol-ent was gently evaporated under a stream of dry N2 to obtainthin film at the bottom of a glass tube. Last traces of solvent

s of Lipids 158 (2009) 46–53 47

were removed by a further 3 h desiccation under high vacuum. Tothe dry samples, 2 ml of a buffer containing 150 mM NaCl, 0.1 mMEDTA, 10 mM Hepes pH 7.4 was added, and vesicles were formed byvortexing the mixture, always keeping the temperature above thegel to liquid-crystalline phase transition temperature of the phos-pholipid. Calcium-containing samples were prepared in the samebuffer, to which a specific volume of calcium-containing bufferwas added in order to obtain the specific phospholipid:calciummolar ratio required. The concentration of ionophore used to allowa rapid equilibration with calcium in the multilamellar vesiclesdid not affect the phase transition of pure DMPS as observed byDSC measurements. Experiments were performed using a MicroCalMC2 calorimeter (MicroCal, Northampton, USA). The final phos-pholipid concentration was 1 mg ml−1. The heating scan rate was60 ◦C h−1. For the measurements at high temperatures (>80 ◦C)the above suspensions were centrifuged at 13,000 rpm in a benchmicrofuge and the pellets were collected into small aluminiumpans. Pans were sealed and scanned in a PerkinElmer DSC-7calorimeter, using a reference pan containing buffer. The heatingrate was 4 ◦C/min. The construction of the partial phase diagramwas based on the heating thermograms of mixtures of DMPS andtrehalose lipid at various trehalose lipid concentrations. The onsetand completion temperatures for each transition peak were plot-ted as a function of the mol fraction of trehalose lipid. These onsetand completion temperatures formed the basis for defining theboundary lines of the partial temperature-composition phase dia-gram.

2.4. X-ray diffraction

Simultaneous small (SAX) and wide (WAX) angle X-ray diffrac-tion measurements were carried out as described previously(Teruel et al., 2004) using a modified Kratky compact camera(MBraum-Graz-Optical Systems, Graz Austria) which employs twocoupled linear position sensitive detectors (PSD, MBraum, Garch-ing, Germany). Nickel-filtered Cu K� X-rays were generated by aPhilips PW3830 X-ray Generator operating at 50 kV and 30 mA.Samples for X-ray diffraction were prepared by mixing 15 mg ofDMPS and the appropriate amount of trehalose lipid in chlo-roform/methanol (8:1). Multilamellar vesicles were formed asdescribed above. After centrifugation at 13,000 rpm, the pelletswere placed in a steel holder, which provided good thermal contactto the Peltier heating unit, with cellophane windows. Typical expo-sure times were 10 min, allowing 10 min prior to the measurementfor temperature equilibration.

2.5. Infrared spectroscopy (FT-IR)

For the infrared measurements multilamellar vesicles wereprepared, as described above, in 50 �l of the same buffer pre-pared in D2O. Samples were placed in between two CaF2 windows(25 mm × 2 mm) separated by 25 �m Teflon spacers and transferredto a Symta cell mount. Infrared spectra were acquired in a Nico-let 6700 Fourier-transform infrared spectrometer (FT-IR) (Madison,WI). Each spectrum was obtained by collecting 256 interferogramswith a nominal resolution of 2 cm−1. The equipment was contin-uously purged with dry air in order to minimize the contributionpeaks of atmospheric water vapor. The sample holder was ther-mostated using a Peltier device (Proteus system from Nicolet).Spectra were collected at 2 ◦C intervals, allowing 5 min equilibrationbetween temperatures. The D2O buffer spectra taken at the same

temperatures were subtracted interactively using either Omnic orGrams (Galactic Industries, Salem, NH) software. In cases whereabsorption bands appeared to be a summation of components,curve fitting procedures were used to obtain the position and con-tribution of the component bands.

48 A. Ortiz et al. / Chemistry and Physic

Fig. 2. DSC heating thermograms for DMPS, and DMPS containing trehalose lipid atd0fa

3

DDic

F4

ifferent concentrations. Molar fraction of trehalose lipid from top to bottom: 0, 0.02,.05, 0.07, 0.10, 0.15, 0.20, 0.30, 0.40 and 0.50. The inset shows the enthalpy changeor the gel to liquid-crystalline phase transition of mixtures of DMPS/trehalose lipidt different molar fractions.

. Results and discussion

The effect of trehalose lipid on the thermotropic transition ofMPS is presented in Fig. 2. In the absence of trehalose lipid,MPS exhibits a single highly cooperative phase transition that

s fully reversible and relatively energetic, that corresponds to thehain melting transition from the gel phase to the liquid-crystalline

ig. 3. Wide angle X-ray diffraction profiles of DMPS system containing different concentr5 ◦C). From top to bottom: pure DMPS, DMPS containing 0.05 mol fraction trehalose lipid

s of Lipids 158 (2009) 46–53

phase. We find that the transition takes place at 36 ◦C with anenthalpy change of 7.2 kcal mol−1, in agreement with previousresults (Marsh, 1990; Lewis and McElhaney, 2000). The presence ofincreasing concentrations of trehalose lipid lowers the phase transi-tion temperature and decreases the cooperativity of this transition.The inset in Fig. 2 shows that the enthalpy change associated withthe gel to liquid-crystalline phase transition of DMPS progressivelydecreases as more trehalose lipid is present in the system, reachingnear 30% of the value of the pure system in the case of the mostconcentrated samples. These results suggest that the hydrophobicmoieties of trehalose lipid incorporate into the phosphatidylser-ine acyl chains palisade, perturbing the acyl chains, reducing thecooperativity of the transition and shifting the phase transitiontemperature to lower values. In the presence of trehalose lipid, thethermograms are fairly asymmetric, suggesting the presence of tre-halose lipid-enriched domains. However, there is not evidence of anew peak or shoulder in the lower part of the thermograms, as itwas the case for mixtures of the biosurfactant with phosphatidyl-cholines and phosphatidylethanolamines (Aranda et al. 2007; Ortizet al., 2008).

X-ray diffraction was used to get information on the structuralproperties of DMPS/trehalose lipid system. Measurements in thewide angle (WAX) region provide information about the packingof the phospholipids acyl chains. Fig. 3 shows the WAX patterncorresponding to pure DMPS and DMPS containing trehalose lipid.At 20 ◦C (Fig. 3, left), the spacing of the WAX reflection for pureDMPS display a value of 4.14 Å, indicating a conventional gel phasein which the acyl chains are packed parallel to the bilayer normalon a regular hexagonal lattice; and at 45 ◦C (Fig. 3, right) pure DMPSshows a very broad component centered at 4.4 Å typical of the disor-dered liquid-crystalline phase, in agreement with previous reports(Hauser et al., 1982; Brandenburg et al., 1999; Teruel et al., 2004).As seen in Fig. 3 the presence of trehalose lipid does not affect thepacking of the DMPS acyl chains below and above the phase tran-sition temperature. Small angle X-ray diffraction (SAX) was used tocheck whether trehalose lipid affects the phase behaviour of DMPS.This technique not only defines the macroscopic structure itself,but also provides the interlamellar repeat distance in the lamellarphase. The largest first order reflection component corresponds tothe interlamellar repeat distance, which is comprised of the bilayerthickness and the thickness of the water layer between bilayers.Fig. 4 shows the SAX diffraction pattern profiles corresponding topure DMPS and DMPS containing trehalose lipid at two different

temperatures. Pure DMPS reveals a diffraction peak with an inter-lamellar repeat distance of 67 Å in the gel phase (20 ◦C, Fig. 4, left).This value decreases above the chain melting temperature to 58 Å(45 ◦C, Fig. 4, right), which is within the range of previous reporteddata (Hauser and Shipley, 1983; Teruel et al., 2004). It is interesting

ation of trehalose lipid in the gel (left, 20 ◦C) and the liquid-crystalline phase (right,, DMPS containing 0.20 mol fraction trehalose lipid.

A. Ortiz et al. / Chemistry and Physics of Lipids 158 (2009) 46–53 49

Fc(t

tbcttoit

gttill(abslpoelbf

Dtpt

Fig. 5. Partial phase diagrams for DMPS in DMPS/trehalose lipid mixtures. Solid (�)

a broadening of the chain melting transition which is shifted tolower temperatures. The presence of a high concentration of tre-halose lipid produces a further broadening and decrease of thephase transition temperature, which is in accordance with the DSC

ig. 4. Small angle X-ray diffraction profiles of DMPS system containing differentoncentration of trehalose lipid in the gel (left, 20 ◦C) and the liquid-crystalline phaseright, 45 ◦C). From top to bottom: pure DMPS, DMPS containing 0.05 mol fractionrehalose lipid, DMPS containing 0.20 mol fraction trehalose lipid.

o note the absence of sharp Bragg reflections and the presence ofroad profiles, in agreement with the early observation that for aontinuous swelling lipid–water system, like DMPS, at high hydra-ion levels, multilayer stacking disorders result in a broadening ofhe small angle diffraction lines (Hauser et al., 1982). The presencef trehalose lipid does not affect the interlamellar repeat distance,ndicating that the biosurfactant does not modify the thickness ofhe bilayer or the thickness of the water layer between bilayers.

DSC and X-ray data were used to construct a partial phase dia-ram for DMPS in mixtures of DMPS and trehalose lipid. Fig. 5 showshat both the solid and the fluid lines display near ideal behaviour,he temperature decreasing as the trehalose lipid concentrationncreases, indicating a good miscibility both in the gel and theiquid-crystalline phases. In all cases the system evolves from aamellar gel phase (G phase) to a lamellar liquid-crystalline phaseF phase) through a coexistence region (G + F), which became widers more trehalose lipid was added to the system. Trehalose lipid haseen found to present fluid immiscibilities in phosphatidylcholineystems (Aranda et al., 2007b). Our results indicate that trehaloseipid is more soluble in phosphatidylserine systems than in phos-hatidylcholine ones, being the phase diagram more similar to thatf mixtures of trehalose lipid and phosphatidylethanolamine (Ortizt al., 2008). This dissimilar effect emphasizes the importance of theipid polar head group in order to determine how the behaviour ofilayer lipid molecules is affected by membrane interacting biosur-actants.

To investigate the effect of trehalose lipid on different parts of theMPS molecule, infrared spectroscopy was used. It has been shown

hat spectroscopic markers emanating from infrared-active groupsresent in the hydrophobic, interfacial, and headgroup domains ofhe lipid bilayer are each differentially sensitive to the thermotropic

and open (©) circles were obtained from the onset and completion temperaturesof the main gel to liquid-crystalline phase transition. The phase designations are asfollows: G, gel phase; F, liquid-crystalline phase.

phase transition observed by calorimetry (Lewis and McElhaney,2000). The CH2 symmetric stretching band near 2850 cm−1 is ofspecial significance because of its sensitivity to changes in themobility and in the conformational disorder of the hydrocarbonchains (Mendelson and Mantsch, 1986). Fig. 6 shows the temper-ature dependence of the frequency at the absorbance maximumof the symmetric CH2 stretching vibration band of the infraredspectra of DMPS and those systems containing trehalose lipid.For pure DMPS in the gel phase the absorption maximum of theCH2 stretching band is observed near 2849 cm−1; on conversionto the liquid-crystalline phase the maximum increases to frequen-cies near 2852.5 cm−1, in agreement with previous results (Lewisand McElhaney, 2000). This frequency increase, which is accom-panied by a broadening of the band (not shown), is a diagnosticsignature of the chain melting phase transitions of hydrated lipidbilayers (Mantsch and McElhaney, 1991). This change is the resultof increased conformational disorder in the hydrocarbon chainsand occurs at the chain melting phase transitions of all paraffiniccompounds (Mendelson and Mantsch, 1986) resulting from theintroduction of a high population of gauche conformers (Lewis andMcElhaney, 2000). Low concentration of trehalose lipid produces

Fig. 6. Temperature dependence of the maximum of the symmetric CH2 stretchingabsorption band exhibited by pure DMPS (�) and DMPS/trehalose lipid mixtures at0.05 (©) and 0.20 (�) molar fraction.

50 A. Ortiz et al. / Chemistry and Physics of Lipids 158 (2009) 46–53

FiD

meosti

cwtbsgttttt1mcptqtipfcD

pcaatMcdssthts1f

ig. 7. Temperature dependence of the maximum of the asymmetric stretch-ng absorption of the head group carboxylate exhibited by pure DMPS (�) andMPS/trehalose lipid mixtures at 0.05 (©) and 0.20 (�) molar fraction.

easurements shown above. An interesting effect is that the pres-nce of a high concentration of trehalose lipid produces an increasef the frequency of the band below and above the phase tran-ition, indicating that in the gel and the liquid crystalline stateshere is an increase in gauche isomers and thus an increase in flu-dity.

The carbonyl stretching region of the infrared spectra of DMPSontains two major bands centered near 1620 cm−1 and 1730 cm−1,hich are attributable to the asymmetric stretching vibrations of

he head group carboxylate and the hydrocarbon chain ester car-onyl groups, respectively (Lewis and McElhaney, 2000). It has beenhown that the asymmetric vibration due to the free carboxylateroup in the hydrophilic region of phosphatidylserine also senseshe phase transition (Dluhy et al., 1983). Fig. 7 shows the tempera-ure dependence of the frequency at the absorbance maximum ofhe carboxylate vibration band of the infrared spectra of DMPS andhose containing trehalose lipid. For pure DMPS in the gel phasehe absorption maximum of the carboxylate band is observed near620 cm−1; on conversion to the liquid-crystalline phase the maxi-um increases to frequencies near 1625 cm−1, both values being

haracteristics of hydrated carboxylates (Casal et al., 1987). Theresence of trehalose lipid produces a broadening and shifting ofhe transition to lower temperatures and an increase of the fre-uency of the band in the gel state. Changes in the frequency ofhis band has been attributed to be the result of a subtle changen the local environment of the head group carboxylate moieties,ossibly because of phase state-induced changes in head group con-ormation and/or orientation (López-García et al., 1993), thus it islear that the presence of trehalose lipid affects the polar moiety ofMPS.

The thermotropic phase change displayed by DMPS is accom-anied by very marked changes in the contours of the esterarbonyl stretching band. As shown before, the properties of thisbsorption band are sensitive to the conformation, hydration state,nd the degree and nature of hydrogen-bonding interactions inhe polar/apolar interfaces of phospholipids bilayers (Lewis and

cElhaney, 1996). It is known that the carbonyl groups of dia-ylphospholipids may be found in lipid vesicles in hydrated andehydrated states, their proportions depending on the physicaltate of the phospholipids bilayer (Blume et al., 1988). Pure DMPSpectra represent a summation of two component bands cen-ered near 1743 cm−1 and 1728 cm−1 (attributed to dehydrated and

ydrated carbonyl groups respectively). The spectra correspondingo pure DMPS and DMPS systems containing trehalose lipid wereubjected to curve fitting to two bands centered at 1743 cm−1 and728 cm−1. These bands were simulated by a Gaussian–Lorentzianunction, and the relative areas of these simulated bands were cal-

Fig. 8. Temperature dependence of the relative proportion of the dehydrated com-ponent of the carbonyl stretching band for pure DMPS (�) and DMPS/trehalose lipidmixtures at 0.05 (©) and 0.20 (�) molar fraction.

culated. Fig. 8 shows the temperature dependence of the percentageof the dehydrated component (1743 cm−1) of the carbonyl stretch-ing band for pure DMPS and DMPS containing trehalose lipid. In thecase of pure DMPS in the gel phase, the higher frequency componentmakes a greater contribution (near 40%) to the overall intensity ofthe carbonyl band than in the liquid-crystalline phase (near 15%).It has been proposed that this decrease in the size of the hydro-gen bonded carbonyl population after formation of the gel phase,could be the result of a phase state-induced decrease in the hydra-tion of the polar/apolar interface (Lewis and McElhaney, 2000). Ascommented above for other infrared absorption bands, the pres-ence of low concentration of trehalose lipid makes the transitionto occur at lower temperatures. Increasing the concentration oftrehalose lipid produces a further decrease of the transition andalso an increase in the proportion of the dehydrated componental all temperatures. The later suggests that trehalose lipid inter-acts with the interfacial region of the bilayer decreasing hydrogenbonding of the phospholipid C O groups with the water moleculesof the hydration layer, and is in line with the dehydrating effectof trehalose lipid observed in phosphatidylcholine (Aranda et al.,2007b) and phosphatidylethanolamine (Ortiz et al., 2008) systems.This effect could be due to the participation of the free hydroxylgroups of trehalose lipid in a high number of hydrogen bonds,which will leave less water molecules available for interacting withDMPS.

It is known that with increasing calcium concentrationthe enthalpy of the gel to liquid-crystalline phase transitionof DMPS decreases. For subsaturating calcium concentration(up to DMPS:calcium of 2:1) an additional transition appearsbetween 44 ◦C and 50 ◦C. For saturating calcium concentration(DMPS:calcium of 1:2) these endotherms are replaced by a broadasymmetric high temperature transition near 155 ◦C (Hauser andShipley, 1984; Hauser, 1991). Fig. 9 shows the effect of trehaloselipid on the thermotropic transition of DMPS in the presence of sub-saturating calcium concentration (DMPS:calcium, 6:1). Pure DMPSshows an asymmetric thermogram with a low enthalpy endotherm,at a temperature similar to that of DMPS in the absence of calcium,and a higher temperature higher enthalpy additional endotherm.The presence of trehalose lipid produces a decrease of the enthalpychange of the overall transition (inset in Fig. 9) and a shift of thetransition to lower temperatures, apparently without a change inthe cooperativity of the transition. Fig. 10 presents the effect of tre-

halose lipid on the thermotropic transition of DMPS in the presenceof saturating calcium concentration (DMPS:calcium, 1:2). As com-mented above, pure DMPS shows an asymmetric endothermic peaknear 155 ◦C, this endotherm is immediately followed by an exhoter-mic peak. These transitions have been assigned to the melting of the

A. Ortiz et al. / Chemistry and Physics of Lipids 158 (2009) 46–53 51

Fig. 9. DSC heating thermograms for DMPS and DMPS containing trehalose lipidafim

cehti

a1Cibgtt(p

Ftt

formed in the presence of calcium, in agreement with previousdata (Hauser and Shipley, 1984). The presence of trehalose lipiddoes not modify the repeat distance and does not affect the struc-tural organization of the calcium/phosphatidylserine complexes. It

t different concentrations, in the presence of calcium (DMPS:calcium, 6:1). Molarraction of trehalose lipid from top to bottom: 0, 0.02, 0.05, 0.10, 0.20, and 0.40. Thenset shows the enthalpy change for the gel to liquid-crystalline phase transition of

ixtures of DMPS/trehalose lipid at different molar fractions.

alcium DMPS complexes (Hauser, 1991). Trehalose lipid shifts thendothermic transition to lower temperatures and broadens theigher temperature exothermic transition. Figs. 9 and 10 indicatehat trehalose lipid is able to perturb the phospholipid palisade evenn the presence of the DMPS–calcium complexes.

The complexes formed by calcium and phosphatidylserine inter-ctions have been characterized by X-ray (Hauser and Shipley,984) and infrared spectroscopic methods (Dluhy et al., 1983;asal et al., 1987). These methods provided evidence that calcium

nteracts strongly forming crystalline calcium–phosphatidylserineilayers, and that calcium interacts mainly with the phosphateroup displacing water hydration. Fig. 11 shows the SAX diffrac-

ion patterns corresponding to pure DMPS and DMPS containingrehalose lipid, in the presence of saturating calcium concentrationDMPS:calcium, 1:2). Pure DMPS is characterized by a diffractionattern in which the sharp Bragg’s reflections are in a ratio 1: 1/2:

ig. 10. DSC heating thermograms for DMPS and DMPS containing trehalose lipid, inhe presence of calcium (DMPS:calcium, 1:2). Molar fraction of trehalose lipid fromop to bottom: 0, 0.05, and 0.20.

Fig. 11. Small angle X-ray diffraction profiles for DMPS and DMPS containing tre-halose lipid at 20 ◦C, in the presence of calcium (DMPS:calcium, 1:2). Molar fractionof trehalose lipid from top to bottom: 0, 0.05, and 0.20.

1/3, indicating a lamellar bilayer structure. The interlamellar repeatdistance of 42 Å is indicative of the dehydrated ordered bilayer

Fig. 12. Infrared spectra of the phosphate symmetric stretching band of pure DMPS(solid lines) and DMPS containing trehalose lipid at 0.20 molar fraction (dashedlines), at 20 ◦C.

5 Physic

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4

aithbttbrthcaWDsmoliwt

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aBM

R

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A

B

2 A. Ortiz et al. / Chemistry and

as been shown that the presence of a series of reflections in theide angle region suggest crystallization of the phosphatidylserine

hains induced by calcium (Hauser and Shipley, 1984), the incorpo-ation of trehalose lipid does not modify these reflections (data nothown).

The effect of chelation of calcium by DMPS on the phosphateroup can be seen by examination of the phosphate symmetrictretching vibration, and this infrared absorption band is pre-ented in Fig. 12. Pure DMPS shows four bands near 1110, 1090,080 and 1065 cm−1, which are characteristic of the anhydrousMPS–calcium complex (Casal et al., 1987). The presence of tre-alose lipid does not change the band pattern, but produces aroadening of the contour of the bands, suggesting an increase inhe overall rates and amplitudes of phosphate head group motions.H2 and carbonyl stretching absorption bands are also broader

n the presence of trehalose lipid (data not shown), indicating anncrease of motional freedom of the phospholipids molecules evenn the presence of calcium.

. Conclusions

The thermotropic and structural properties of mixtures of DMPSnd a trehalose lipid bacterial biosurfactant have been exam-ned to establish the extent of intermolecular interaction betweenwo types of molecules. Our DSC and X-ray diffraction resultsave evidenced that trehalose lipid incorporates into DMPS mem-rane and perturbs its thermotropic gel to liquid-crystalline phaseransition, decreasing the cooperativity and lowering the transi-ion temperature. Trehalose lipid does not affect the macroscopicilayer organization of DMPS; it does not change the interlamellarepeat distance; and it shows good miscibility both in the gel andhe liquid-crystalline phases. Infrared experiments show that tre-alose lipid increases the fluidity of the phosphatidylserine acylhains, changes the local environment of the polar head group,nd decreases the hydration of the interfacial region of the bilayer.hen trehalose lipid is incorporated into the crystalline dehydrated

MPS/calcium complexes, it is able to affect the thermotropic tran-ition and to increase the motional freedom of the phospholipidsolecules. Given the critical structural and functional importance

f phosphatidylserine in membranes, and the wide variety of bio-ogical actions that are exerted by trehalose lipid, the observednteractions between trehalose lipid and DMPS presented in this

ork may help to explain the molecular interactions underlyinghese effects.

cknowledgements

This work was financially supported by Project CTQ2007-66244nd PET2005-0396 from Ministerio de Educación y Ciencia; andIO-QMC-06/01-001 from Comunidad Autónoma de la Region deurcia, Spain.

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