Melting of Single Lipid Components in Binary Lipid Mixtures: A Comparison between FTIR Spectroscopy, DSC and Monte Carlo Simulations

0.41 MB·English

by  M. Fidorra

Melting of Single Lipid Components in Binary
Lipid Mixtures: A Comparison between FTIR
Spectroscopy, DSC and Monte Carlo Simula-
tions
8
0
0
2
M. Fidorra†,‡, T. Heimburg† and H.M. Seeger†,⋆,∗
n
a
J
†TheNielsBohrInstitute,UniversityofCopenhagen,Copenhagen,Denmark
3
‡MEMPHYS-CenterofBiomembranePhysics,DepartmentofPhysics,

] UniversityofSouthernDenmark,Odense,Denmark
h ⋆ CNR-INFMNationalResearchCenteron’nanoStructuresandbioSystemsatSurfacesS3’,
p
Modena,Italy
-
o
i Monte Carlo (MC) Simulations, Differential Scanning Calorimetry (DSC) and Fourier Transform InfraRed (FTIR)
b
spectroscopy were used to study the melting behavior of single lipid components in two-component membranes of 1,2-
.
s Dimyristoyl-D54-sn-Glycero-3-Phosphocholine (DMPC-d54) and 1,2-Distearoyl-sn-Glycero-3-Phosphocholine (DSPC).
c
i MicroscopicinformationonthetemperaturedependentmeltingofthesinglelipidspeciescouldbeinvestigatedusingFTIR.
s
The microscopic behavior measured could be well described by the results from the MC simulations. These simulations
y
h alsoallowedtocalculateheatcapacityprofilesasdeterminedwithDSC.Theseonesprovidemacroscopicinformationabout
p melting enthalpiesand entropychangeswhich are not accessible with FTIR. Therefore, the MC simulationsallowed us to
[ linkthetwodifferentexperimentalapproachesofFTIRandDSC.

2
v
4 keywords:domainformation;phaseseparation;FTIR;MCsimulation;DSC;meltingofsinglelipidcomponents
6
0 Introduction
ture also called gel) to a liquid disordered (ld; or named as
0
fluid)phasearepresentinartificialandbiologicalmembranes
.
2 Biologicalmembranesdisplaya complexlipid composition. (13, 14, 15, 16, 17, 18). In the presence of cholesterolalso
1
Thereasonforthebigvarietyinlipidspeciesisstillnotclear. liquidorderedphasesdevelop(19). Consideringsinglelipid
7
Ithasbeenshownformanydifferentbacteriathattheirlipid membranes one finds that a higher degree of saturation or
0
: synthesisdependsonphysicalparameterssuchasgrowthtem- longerfattyacidsresultinhighermeltingtemperatures(20).
v
peratureorhydrostaticpressure(1,2,3,4,5,6,7,8,9,10,11, Therefore,onewouldexpectachangeofthemeltingbehav-
i
X regard also the citations given therein) . It could be shown ior of biological membranes in dependence of growth tem-
r thatthemembranesofbacteriagrownathighertemperatures peratureorhydrostaticpressureandtherewithindependence
a
containmoresaturatedfattyacidchainsandchainstendtobe of lipid synthesis. Indeed, this has been demonstrated for
longer (1, 2, 3, 4, 5, 6, 7, 8, 11, see also citations therein). theprominentexampleoftheinnermembraneofEscherichia
Higherhydrostaticpressureinducesanincreasedsynthesisof coli(21).
unsaturatedlipids(9, 10), whichhasalsobeenfoundforbi-
Thedependenceoflipidsynthesisbyouterphysicalparam-
ological membranesfrom several deep-sea fish tissues (12).
eterssuchastemperatureorhydrostaticpressurewastermed
Thisadaptionhasaninfluenceonthephysicalbehaviorofa
“homeoviscousadaption”(22,23). Theunderlyingideawas
biologicalmembranesuchasthemeltingtransitionbehavior.
thatforapropercellfunctionthemembraneneedstoprovide
Melting transitions from a solid ordered (so; in the litera-
acertainviscosity. Inthiscontextthewidelyusedterm“flu-
idity”wasintroducedintheliterature. Itshould,however,be
Abbreviations: DSC,differential scanningcalorimetry; FTIR,fourier
transform infrared spectroscopy; MC simulation, Monte Carlo simula- noted that this term lacks a clear definition. It was also ar-
tion; DMPC, 1,2-dimyristoyl-sn-glycero-3-phosphocholine; DMPC-d54, gued againsta role of “fluidity” (24). An alternativerole of
1,2-dimyristoyl-d54-sn-glycero-3-phosphocholine; DSPC, 1,2-distearoyl- the adaption of lipid synthesis on outer physical parameters
sn-glycero-3-phosphocholine
∗ Corresponding author: [email protected], Via G. Campi istoensureacontroloftheheterogeneityofthelateralmem-
213/A,41100Modena,Italy branestructure(25). Thisideahasobtainedspecialattention
1
2 Fidorraetal.
withinthediscussionabout“rafts”(26). 19−20◦C (49,50).
Artificial lipid membranes consisting of one, two or three Forthedescriptionof phaseseparationin lipidmembranes
lipid components are suitable systems to study the general various theoretical approaches have been applied. This in-
physicalbehavioroflipidphasetransitionsinmorecomplex cludesidealandregularsolutiontheory,meanfield,butalso
lipidsystems. Inthephasecoexistenceregimedomainswith numericalapproachessuchasMonteCarlo(MC)simulations
asizerangingfromafewnanometerstoseveralmicrometers (48, 51, 52, 53, 54). Ideal and regular solution theories al-
forminmodelsystemsand canbe observedby a largevari- low the understandingof phase diagrams. In the ideal solu-
ety of experimentaltechniques, such as Fluorescence Reso- tiontheorycompletemiscibilityinthesolidorderedandliq-
nanceEnergyTransfer,AtomicForce MicroscopyandCon- uiddisorderedphaseisassumed. Theregularsolutiontheory
focalFluorescenceMicroscopy(27,28,29,30,31). takesastepfurtherandregardsanon-idealmixinginthesolid
ordered phase. These considerations allow the construction
Already since the early 1970sthe idea that domainforma-
ofphasediagrams.Phasediagramsareusedtomapthephase
tionprocessescaninfluenceproteinactivityandfunctionwas
behavior of lipid membranesin dependenceof e.g. temper-
expressed(25, 32, 33). Inseveralpublicationsthe existence
ature and lipid composition. The theoretically derivedlever
ofdomainshavebeenshowntoprovideamechanismtocon-
rule providesthe possibility to determine the ratio of disor-
trolbiochemicalreactioncascades(34, 35, 36, 37, 38). Ev-
dered and ordered lipids in dependence of temperature and
idence that lipid phase transitions and domain coexistence
lipidcompositionofbinarylipidmixturesandcanalsobeap-
trigger enzyme activity has been found for various systems.
pliedtoexperimentallymeasuredphasediagrams(48). Ideal
Forexample,theenzymephospholipaseA2whichhydrolyzes
or regular solution theory are, however, based on simplify-
the sn-2 bond of phospholipids, producing a free fatty acid
ingassumptions. Thisdoesnotonlyincludeanidealmixing
and a lysolipid, is strongestin activity when the lipid mem-
intheliquidphase,butalsotheassumptionofphasesepara-
braneisinthelipidphasetransitionregime(39,40,41). The
tionunderallconditionsinthemeltingregime.Thisis,how-
activityofthespecialclassofphospholipaseA2typeIIAhas
ever, not the case (55). Further, fluctuations which are en-
beendemonstratedto berelatedto anenrichmentofanionic
hancedin themeltingtransitionregimearepresent(56, 57).
lipidsintold domains(42). Inthiscase,adifferentpartition
This is where MC simulations analyzing simple models of
behavior of the anionic lipids into ld and lo domains led to
lipidchainmeltingenter. Theyallowtheconsiderationofin-
a localconcentrationhigherthanthe thresholdvalueforen-
teractions between different lipid species and varying chain
zymeactivity.Changesintheopeningtimesandprobabilities
state. The formation of lipid domains and phases and the
ofcalciumchannelsreconstitutedinPOPE:POPCmembrane
consideration of fluctuations is a direct consequence of the
ofvariousratioshavebeeninterpretedtobeduetothecoex-
thermodynamicsofthesesystemswhichisincludedinthese
istenceofsolidorderedandliquiddisorderedphases(43). A
models.Therefore,thesesimulationscandescribelipidmem-
furtherexampleistheproteinkinaseC(PKC)whichmodifies
branepropertiesinthevicinityoflipidphasetransitions,such
proteinsbychemicallyaddingphosphategroups. Ithasbeen
aschangesinmembranepermeability(58),fluctuationsinen-
discussed that lateral heterogeneities of the lipid membrane
thalpy(57)anddiffusionprocesses(59,60)well.
control the activation of this enzyme (44, 45). In the latter
study it has also been pointed out that domains enriched in In this study we determined the amount of the single lipid
dioleoylglycerollipidsplayacrucialrole,butalsoalinearre- speciesinthedifferentdomainsofbinaryDMPCandDSPC
lationshipbetweenPKCactivityandphosphatidylserinecon- mixturesbymeansofMCsimulationsandcomparedthere-
tenthasbeenfound(46). Furthercommunicationscanbere- sultsobtainedtoexperimentaldatameasuredbyFourierTrans-
trievedwhereaconnectionbetweenenzymeactivityandthe formInfraRedspectroscopy(FTIR).
presenceofspecificlipidsandstructurearemade.Thisise.g. Among other techniquesFTIR is well suited to investigate
alsothecaseforthecalciumATPase(47). themeltingbehavioroflipidmembranes.Inthesestudiesthe
The publications cited above are only a small fraction of temperaturedependenceofvariousvibrationalmodesdueto
manyin thatrespect, buttheyalreadyshow thatit is impor- structuralchangesprovideaversatiletoolinfollowingphase
tantnotonlyto study domainformation,butalso to investi- transitions(61).Articleshavebeenpublishedonexperiments
gatethefractionofthesinglelipidspeciesinlipidmembranes in which FTIR was used to measure nanoscale domain size
showinglateralinhomogeneityduetophaseseparation. The (62)andchainorderparameters(63).
singlelipidspecieswilloccurindifferentamountsinthedif- InourstudythehydrogenatomsofDMPCwerereplacedby
ferent domains that form in a phase transition regime (48). deuteriumatoms. DuetothisthemeltingoftheDMPClipid
Theworkpresentedhereconcentratesonthisforthecaseof chains could be separated from the DSPC lipid chain melt-
abinarylipidmixturecomposedofDMPCandDSPC.These ing,astheCH2andCD2symmetricandasymmetricstretch
twophospholipidsdisplaymeltingtransitioneventswithtran- vibrations differ in frequency due to the heavier deuterium
sition midpoint temperatures of 23.6◦C or 54.4◦C, respec- atoms.Thisallowedustocomparethemeltingofsinglecom-
tively(20).InourstudyweusedadeuteratedDMPCderivate ponentswith MC simulation results which contain informa-
whose phase transition midpoint temperature lies at around tionofthedisorderedchainratioofsinglelipidcomponents.
MeltingofSingleLipidComponents 3
MCsimulationsareabletodescribeexperimentalheatcapac- many) equipped with a mercury-cadmium-telluride (MCT)
ity profileswell. Therefore,theyprovidea meansto couple detector. The spectrometer was flushed with nitrogen start-
thetwodifferentexperimentalapproaches. ing about 30 minutes before the experiments. Temperature
The experimental absorption spectra were evaluated using controlwasdonebyaselfbuiltsampleholderconnectedtoa
twomethods.Oneofthemethodsprobedthetemperaturede- waterbath(DC30-K20,ThermoHaake,Karlsruhe,Germany).
pendenceofthepositionoftheabsorptionmaximaoftheanti- Thescanrateforthetemperaturerampwassetto20◦C/h.The
symmetricCD2 andsymmetricCH2 stretchvibrations. The temperatureinthesamplecorrespondingtoeachtemperature
other one evaluated peak area changes of difference spectra point set with the water bath was appointed in a reference
(DifferenceSpectramethod). Simulationresultsonthefrac- scan. Backgroundspectrawereacquiredateachtemperature
tion of disordered chains of the single lipid species agreed point after lifting the sample compartment out of the beam
well with the data found using the latter method. FTIR al- path. We were interested in the IR absorption due to the
lowstodistinguishthemeltingofthesinglelipidspeciesand symmetric and asymmetric stretch vibrational modes of the
itprobesthemicroscopicbehavior. DSCprobesthebulkbe- CH2andCD2groupsofthelipidfattyacidchains.Example
haviorandmacroscopicobservables.TheMonteCarlosimu- absorption maxima of the CH2 symmetric and asymmetric
lationswereusedasanapproachtocombinetheinformations stretch vibrations at different temperatures are displayed in
ofthetwoexperimentaltechniques. DSC,MCandFTIRare theleftpaneloffig.2.
a potent combination to investigate the melting behavior of Two methods to analyze the FTIR spectroscopy data were
lipidmembranes. used. In a first approach FTIR spectroscopy data was pro-
cessedwithaself-writtenpluginfortheIGORsoftwarepack-
age(WaveMetrics,LakeOswego,OR,USA).Theabsorption
Materials and Methods
peakswerefittedwithapolynomialfunction. Thenthepeak
positionwasdeterminedbycalculatingtherootofthefit.
SamplePreparation:
ForthesecondprotocolthepeakareaoftheCH2orrespec-
Thelipids1,2-dimyristoyl-d54-sn-glycero-3-phosphocholine
tivelyoftheCD2stretchvibrationswasnormalizedtoavalue
(DMPC-d54)and1,2-distearoyl-sn-glycero-3-phosphocholine
of 1×cm−1. A referencespectrum was chosen whichwas
(DSPC)werepurchasedfromAvantiPolarLipids(Alabaster,
taken at a temperature below the melting transition regime.
USA). Samples were prepared from lipid stock solution in
This spectrum was subtracted from all other spectra which
chloroform. These were prepared in chloroform from lipid
were recorded at different temperatures (see the right panel
powder as delivered from the vendor without further purifi-
of fig. 2). Then the peak area located at the positive axis of
cation. To prepare lipid samples in buffer, chloroformfrom
ordinateswascalculatedandplottedasafunctionoftemper-
thedesiredamountoflipidstocksolutionmixturewasevap-
ature. Therewasnodifferenceinsurveyingboth,symmetric
orated under a stream of nitrogen and the sample was kept
andantisymmetricmodesorthesingleones.Further,weshall
under vacuum for several hours afterwards. After this pro-
callthismethodtheDifferenceSpectramethod.
cedure the dried lipid film was hydrated with preheated ul-
trapurewateratatemperatureof60◦C,whichishigherthan
the melting temperature of the higher melting lipid compo- Model:
nentDSPC(54.4◦ C,(20))andkeptstirringatthistempera- We have already applied an Ising-like model to the study
tureforabout30minutes.Incalorimetricexperimentsalipid of diffusion (59), fluctuation (57) and relaxation properties
concentration of 10mM was used. The Fourier Transform (64) of DMPC:DSPC lipid mixtures. In this paper we used
Infraredspectroscopymeasurementsrequiredconcentrations this model to describe FTIR measurements on mixtures of
of 65mM. In order to facilitate lipid membrane hydration DMPC-d54andDSPC andlinkedthese withDSCmeasure-
under these high concentrationsthe samples were subjected ments.
toseveralfreeze-thawcycles. Themodelhasbeenpresentedindetailelsewhere(54, 59).
Inshort, the modelisbasedonthe assumptionthatthelipid
DSCexperiments: chain states can be describedby assigningeither an ordered
DifferentialScanningCalorimetry(DSC)experimentswere ordisorderedstatetothefattyacidlipidchains. Lipidchains
performedonaVP-DSC(MicroCal,Northampton,MA,USA) arearrangedonatriangularlatticeandonlynearestneighbor
withascanrateof15◦C/h(DMPC-d54:DSPC50 : 50)orof interactionsareincluded. Lipidchainsmightchangethede-
5◦C/h (DMPC-d54)per hourin upscan direction. The sam- gree of order or their positions during the simulation. The
pleswereequilibratedatthestartingtemperaturefor30min- model is evaluated with Monte Carlo (MC) simulations in
utes. which the free energy difference of an old and a trial con-
figurationissufficientfordecisionmakingoftheacceptance
FourierTransformInfraRedspectroscopyexperiments: ofanewconfiguration.Thefreeenergyofthesystemcanbe
FourierTransformInfraRed(FTIR)spectroscopyexperiments divided into a configuration dependent and an independent
wereperformedonaVertex70(BrukerOptics,Bremen,Ger- part. Theconfigurationdependentcontributionequals(54):
4 Fidorraetal.
MCsimulationswereusedtoevaluateasimplemodelbased
on the assumption of two lipid chain states, the considera-
∆G(S) = N1d(∆H1−T∆S1)+N2d(∆H2−∆S2) tionofnearestneighborinteractionsonlyandtheapplication
+ Nodωod+Nooωoo+Nodωod (1) of a hexagonallattice. This model has already been shown
11 11 12 12 12 12
+ N1d2dω1d2d+N1d2oω1d2o+N2o2dω2o2d, todescribemeltingprocessesinDMPC:DSPCmixturessuc-
cessfully(54,57,59).Theevaluationneedsthedetermination
where∆H and∆S aretheenthalpyandrespectiveentropy oftenparameters. Thesearetwiceenthalpyandentropydif-
i i
differences of the disordered to the ordered state of DMPC ferencesbetweenthetwodisorderedandorderedchainstates
(i = 1) and DSPC (i = 2) lipid chains. ωmn are inter- and six unlike nearestneighborinteraction parameters. The
ij
action parameters of unlike nearest neighborsand Nmn the enthalpy and entropy changes can be deduced easily from
ij
corresponding number of the unlike nearest neighbor con- heatcapacitymeasurementsofthesingleonecomponentsys-
tacts with (i 6= j) and (m 6= n). m and n denote the state tems. Two interaction parameters describe the cooperativ-
ofachainwhichiseitherordered(o) ordisordered(d). En- ity, meaning the width of these transitions (65, 66). The
thalpy, entropy and nearest neighbor interaction parameters fourparametersremainingneedtobededucedfromcompar-
canbeobtainedfromexperimentallydeterminedheatcapac- isons with cp-profiles obtained for different lipid composi-
ity profiles. Previously, we have determinedthe parameters tions. Previously, we have determined the parameters for a
neededfortheDMPC:DSPCsystem(59).Inthispaper,how- DMPC:DSPC system (59). In this study, however, we ex-
ever,FTIRexperimentswereperformedwithDMPC-d54and changed the DMPC lipids with deuterated DMPC (DMPC-
DSPCmixtures. Inthefollowingitwasassumedthattheun- d54)whichgivesheatcapacityprofileswhicharebroadened
like species interaction parameters did not change from the and shifted by −3.9◦C to lower temperatures in compari-
systemDMPC:DSPCtoDMPC-d54:DSPC.Theheatcapac- son to the nondeuterated DMPC (data not shown and see
ityprofileofthepureDMPC-d54wasmeasuredtoobtainits (49,50)). Thismeansthatwehadtodeterminetheenthalpy
enthalpy and entropy changes during the melting transition andentropychangesandthecooperativityparameterofpure
and the cooperativity parameter ωod (data not shown). The DMPC-d54.Inprincipleusingthismodeloneneedstoreestab-
11
remaining parameters were taken from a recent publication lishallparametersforanybinarylipidsystem. Inthiswork,
(59). Allvaluesarelistedintable 1. however,we assumedthatthe unlikenearestneighborinter-
action parametersdo not changeby the replacingof DMPC
with its deuteratedanalogand we adoptedthem fromprevi-
Tm,1=19.7 ◦C ω1o1d=1267 J/mol ous simulations of the nondeuterated DMPC:DSPC system
Tm,2=54.8 ◦C ω2o2d=1474 J/mol (59). To validate this, DSC measurements on an equimolar
∆H1=14500 J/mol ω1o2o=607 J/mol DMPC:DSPC mixture were performed and they were com-
∆S1=49.5 J/(mol◦C) ω1d2d=251 J/mol pared to results simulated. We found that the values calcu-
∆H2=25370 J/mol ω1o2d=1548 J/mol lateddescribethemeltingprofilemeasuredwell(see fig.1).
∆S2=77.36 J/(mol◦C) ω1d2o=1716 J/mol Therefore,we were convincedthatit was feasible to further
usethemodelwiththeinteractionparametersgivenintable1.
Table1:ParametervaluesusedintheMonteCarlosimulations.Theywere
Thefurtherscopeofthispaperwastocomparethetemper-
determinedfromexperimentallydeterminedheatcapacityprofiles(59). All
numbersaregivenperlipidchain.Theindicesoanddstandfororderedand aturedependenceofthesimulateddisorderedchainratiosof
disordered,respectively. 1and2indexDMPC-d54andDSPC. DMPC-d54andDSPC lipidswiththe experimentalanalysis
of the melting processes using FTIR spectroscopy. This in-
directly allowed us to compare the experimentalresults ob-
Thesimulationsweredoneonmatrixsizes60×60(DMPC-
tainedwithDSCandFTIR.
d54:DSPC100:0,70:30,60:40,50:50,40:60,30:70),100×
In FTIR spectroscopy the absorption of infrared light due
100(90:10,80:20,20:80,10:90)and350×350(DSPC)(54).
to vibrational degrees of freedom in the sample is studied.
Thesystemwasequilibratedfor5000oraroundtheheatca-
Inlipidsuspensionsdifferentvibrationalmodesofthelipids
pacitymaximafor10000MCcycles. Simulationswerecon-
contribute to the absorption spectrum. Prominent examples
ductedover50000or100000MCcycles,respectively.
usedinourstudyareCH2orCD2(inthecaseofthedeuter-
ated DMPC lipids) stretch vibrations of the fatty acid lipid
Results chains. Theshapeoftheabsorptionbandistemperaturede-
pendent as can be seen in the left panel of fig. 2. Example
In this paper we discuss melting processes in binary lipid spectra of the symmetric and asymmetric CH2 vibrational
mixtures. Thereby, we focus on the melting of the two sin- spectra are given for different temperatures of a mixture of
glecomponentsindependently.ExperimentalFTIRandDSC DMPC-d54:DSPC40:60. Deuteriumatomsareheavierthan
measurements were accompanied by numerical simulations hydrogenatoms. ThisleadstoashiftoftheCD2 stretchvi-
andananalysisofthecorrespondingphasediagram. brationstolowerwavenumbersandallowstodistinguishthe
MeltingofSingleLipidComponents 5
twodifferentlipidspeciesinabinarylipidmixture. butalsoto usea DifferenceSpectramethod too. Furtherwe
studiedthepeakareaofthesedifferencespectraasafunction
oftemperature.
ComparisonofFTIR Results andMonteCarlo Simula- Intheleftpaneloffig.2examplespectraoftheCH2stretch
tions vibrationsaregivenasafunctionoftemperatureofaDMPC-
d54:DSPC40:60mixture. Normalizingtheareaofthespec-
trum to 1×cm−1 and defininga referencespectrum which
In an investigation of structural changes in cytochrome c
liesatatemperaturebelowthemeltingtransitionregime,dif-
Heimburg and Marsh (67) focused on temperature depen-
ferencespectrawerecalculated.Representativeexamplesare
dent changes of difference spectra. This was motivated by
displayed in the right panel of fig. 2. To follow the melt-
theideathatthecorrespondingamideIbandisaconvolution
ing transition the change in difference spectra peak area of
ofavarietyofdifferentbands. Thetemperaturedependence
of each of the single bands differs and a complex tempera- the CH2 or the CD2 symmetric, asymmetricor bothvibra-
tionalmodescanbemonitored.Ourexperimentsshowedthat
turedependentbehaviorispresent.Therefore,wedecidednot
there is no difference between these three approaches(data
onlytoinvestigatethetemperaturedependenceoftheabsorp-
notshown).
tionmaximaasdoneintheliteraturepreviously(28,68,69),
Thetemperaturedependentevolutionofthedifferencespec-
tra peak areas monitored are given in fig. 3 as solid curves
for four different DMPC-d54:DSPC mixtures: (A) 70:30,
(B) 50:50, (C) 40:60and (D) 30:70. The panels on the left
side depict the results related to the deuterated DMPC-d54
lipidsandtherightonesgivethedatabelongingtotheDSPC
lipids. The data obtainedis also comparedto simulationre-
sultsoftheratioofdisorderedlipidchainsforeachofthesin-
glespecies. Infig.3thesearegivenasopencircles(DMPC-
d54) or squares (DSPC). In all instances we found an in-
crease of the area with higher temperatures. In all cases a
goodagreementbetweenthesimulatedandmeasuredvalues
ispresent.Thedeviationsofthedatasimulatedfromthemea-
suredonesaresmallerinthecasesoftheDMPC-d54lipids.
This might be due to the fact that the determination of the
model parameters needed is imperfect. More extended and
time-consumingsimulationsmightimprovethedata,too.
As alreadymentionedabove, previouslymelting processes
Figure1: Enthalpyandentropychangesandcooperativ- oflipidmembranesinvestigatedbyFTIRwereanalyzedmon-
ityparametersofthesingleDMPC-d54andDSPClipid itoringthetemperaturedependenceofthepositionoftheab-
membranesweredeterminedfromheatcapacityprofiles. sorption maximum of either the symmetric or asymmetric
The remaining four unlike species nearest neighbor in- stretch vibrationsof CH2 and CD2. We analyzed the tem-
teractionparametersofDMPC-d54:DSPCmixtureswere perature dependence of the absorption maxima for several
assumed to equal the ones of a DMPC:DSPC system DMPC-d54:DSPC mixtures as earlier done by Leidy et al.
(59).SimulatinganequimolarmixtureofDMPC-d54and (28),too.Theresultsobtainedaregivenbythedashedcurves
DSPCgivesameltingprofile(markers)whichdescribes infig.3.
wellthemeasuredheatcapacityprofile(solidcurve).Be- We confirmedwhat has been reportedby Leidy et al. (28)
low three representative snapshots from simulations of previously. There are instances when the positions of the
thesamemixturewithamatrixsizeof80×80lipidchains maximaofthesymmetricstretchvibrationsoftheDSPClipids
areshown.Theredandgreencolorsrepresentorderedor donotincreasewithhighertemperatures,butthatforcertain
disordered lipid chains, respectively. The lighter colors lipid mixture fractions they decrease, approach a minimum
correspondtotheDMPC-d54lipidchainsandthedarker and then they start to become larger again. The minimum
onestotheDSPClipidchains.Thesnapshotsweretaken wavenumberisin allcases lowerthanthe wavenumbersde-
atthreedifferenttemperatures. Atatemperatureof15◦ terminedatlowertemperatures.Thisbehaviorisindisagree-
Cthemembraneisinthesolidorderedphasewhileata ment with the evolution of the difference spectra peak area
temperatureof55◦Citisintheliquiddisorderedphase. andtheMCsimulationresults.
In both cases lipids do not mix ideally, but lateral het- Itiswellknownthatmacroscopic,butalsolocalfluctuations
ereogeneities are present. Two macroscopic phases are ofvariousmembranepropertiesareenhancedinthevicinity
presentatatemperatureof33◦C. oflipidmembranemeltingtransitions(56,57). Thedetermi-
6 Fidorraetal.
Figure2: TheIRabsorptionspectrumcontainsdifferentabsorptionmaximaoriginatingfromvaryingvibrations.
In the left panelthe asymmetric and the symmetric CH2 stretch vibrationsas measured in a sample of DMPC-
d54:DSPC40:60 at different temperatures are displayed. The absorption maxima of these are temperature de-
pendentandliearound2852cm−1(symmetric)andat2925cm−1 (asymmetric). Theasymmetricandsymmetric
CD2 stretch vibrationsareshifted tolower wavenumbersdueto theheavierdeuteriumatoms(datanotshown).
Inpreviousstudiesfromotherlabsthemeltingprocesseswereinvestigatedfollowingthetemperaturedependence
of the absorption maxima. In this study, however, the melting process was also studied by defining a reference
spectraandsubtractingthisonefromallotherspectrawhereineachcasestheareawasnormalizedto1×cm−1
(DifferenceSpectramethod). Exampleresultsofthedifferencespectraoftheleftpanelaredisplayedintheright
panel.Asameasureoftheprogressofthemeltingprocesstheareaofoneofthepeakswastaken.
nationofthederivativesofthedataofthecurvesfromfig.3 Themeltingtransitionofthesinglelipidcomponentshappens
as done by Leidy and collaborators(28) provides a measure overabroadtemperatureregime.
ofthestrengthoffluctuationswithrespecttothesinglelipid
components.Infig.4thesederivativesareshownforthedata
ThePhaseDiagram
simulated. Thehigherthevalueofthederivativethestronger
thefluctuations.DMPC-d54meltsatlowertemperaturesthan
DSPC. This is why the correspondingderivativesin the left Phase diagrams are generally constructed to easily access
panelsoffig.4havemaximaatlowertemperatures.Thistem- thephaseseparationbehaviorofcomplexlipidmixtures(48).
perature, however, depends on the molar fraction of DSPC In our case a phase diagram depending on lipid composi-
lipids and is higher with an increased DSPC lipid fraction. tion and temperaturewas constructed. A phenomenological
This is similar for the DSPC lipids only with the difference methodtoconstructtheseisinaligningtangentsonthelower
that the presence of DMPC-d54 lowers the temperatures at and uppertemperaturelimits of heat capacity profiles. This
whichfluctuationsoftheDSPClipidchainsarethestrongest. resultsinthe constructionofasolidusanda liquidusline as
Inthiscontextitshouldbenotedthatthecomponentwhichis indicatedbyopensquaresandthegreycurvesinfig.5. Ifthe
intheminorityalwaysdisplaystwomaxima.Oneofthemax- lipid composition is known, the physical state of the mem-
imais a localmaximumatwhichfluctuationsareenhanced, brane can be determined. The data obtainedfromthe lower
but they are lower than at the global maximum. The local temperature limit defines the solidus line, whereas the one
maximumisfoundatatemperatureatwhichtheothercom- fromtheuppertemperaturelimitgivestheliquidusline. Be-
ponent displays its strongest fluctuations. At an equimolar lowthesoliduslinethelipidmembraneisinthesolidordered
ratio of DMPC-d54:DSPC this is true for bothcomponents. phase, while abovethe liquidusline it is in the liquiddisor-
dered phase. Therewith, the liquidus and the solidus lines
MeltingofSingleLipidComponents 7
Figure3: The FTIR resultsare comparedwith the Monte Carlo simulations. The dataon fourdifferentDMPC-
d54:DSPCratios70:30(A),50:50(B),40:60(C)and30:70(D)aredisplayed.Ineachpanelthedisorderedchain
ratios obtained from the simulations (open circles (DMPC-d54) and open squares (DSPC)) are compared with
thechangesofthepeakarea(solidcurves)andtheevolutionofthepositionoftheabsorptionmaximum(dashed
curves). InthelattercasetheantisymmetricCD2 (DMPC-d54)andthesymmetric CH2 stretchvibrationswere
followed. ThepanelsontheleftsidedepictthemeltingprocessofthedeuteratedDMPC-d54lipidsandtheright
onestheDSPClipidmelting. TheMonteCarlosimulationsallowtocombineboththeFTIRandthecalorimetric
results. Theyprovideameantorelatethemacroscopicwiththemicroscopicbehaviorofthebinarylipidmembrane
melting.
definethephasecoexistenceregimetoo. Thethreesnapshots DSPC hasbeenclaimedto beofsecondorderandtherefore
infig.1provideavisualimpressionofthis. Attemperatures the interpretationof the phase diagram has to be taken with
of15◦ Cand55◦ Cthelipidmembraneisineitherthesolid care(55).
ordered or liquid disordered phase, respectively. It should,
With the determination of the derivatives of the curves in
however, be mentioned that the mixing of the two lipids is
fig. 3 we were able to deducethe temperaturesat whichthe
in neither of the two cases ideal. The single lipid species
fluctuations of the single components were enhanced. The
cluster. At a temperature of 33◦ C the two phases coexist
lower temperature melting component, in this case DMPC-
anda macroscopicphaseseparationis present. Inan earlier
d54, has its maximum at lower temperatures than the one
publicationwecouldshowthatdiscussingdomainformation
whichmeltsathighertemperatures(DSPC).Thesetempera-
processesinlipidmembranesoneneedstodistinguishmicro-
tures,however,arehigherorrespectivelylowerthanthemelt-
scopic and macroscopic phase separation and local fluctua-
ingtemperaturesofthe puresinglecomponents. Thevalues
tions in chain state enter the physicalpicture. These fluctu-
aregiveninfig.5. Opencirclesrefertothevaluesoriginating
ations are enhanced at domain or phase boundaries. Small
fromthesimulationswhilethecrossescomefromtheexperi-
domainsarealsosubjecttostrongfluctuations(57). Analyz-
ments. Thetemperaturepointsdeterminedusingthetwodif-
ingphasediagramsoneshouldnotethatthesephysicaleffects
ferentmethodstoanalyzetheFTIRabsorptionspectraarethe
areingeneralnotconsideredinitsinterpretation. Further,it
same(datanotshown). Theexperimentalandthesimulated
hasbeenpointedoutthattheconstructionofaphasediagram
dataagreewell.
isstrictlyspeakingonlyvalidifthetransitionisoffirst-order
Thesimulationsfurtherallowedustocomparethetempera-
(70). The transition of binary lipid mixtures of DMPC and
turesatwhichtheheatcapacitycurvesareinamaximumand
8 Fidorraetal.
Figure4: ThederivativesofthecurvesofthefractionofdisorderedDMPC-d54andDSPClipidchainsasshown
infig.3aredisplayedinthisfigure.Indetailthesearethemixtures70:30(A),50:50(B),40:60(C)and30:70(D).
ThederivativesbelongingtoDMPC-d54areindicatedbyopencircles,whiletheonesofDSPCbyopensquares.
Amaximuminthederivativemeansthatatthistemperaturefluctuationsareenhanced.
the temperatures at which the derivatives of the disordered approaches.
chainratiosdisplaytheirmaxima. We foundthatthesetem-
peraturesequaleach other (data not shown). In some cases
Discussion
onlyoneheatcapacitymaximumisresolvable. Theanalysis
ofthesinglecomponents,however,allowstodistinguishthe
maximaofthemeltingofsinglecomponentsinallinstances. Inthispaperwepresentedacombinednumericalandexperi-
The maxima of the component which is in a majority then mentalstudyexploringmeltingprocessesinbinarylipidmix-
agreeswiththemaximumoftheheatcapacitycurve. tures. Indetail,wefocusedonthemeltingofthesinglelipid
components DMPC-d54 and DSPC independently. Monte
In total, we showed a combined FTIR and MC simulation Carlo simulations linked FTIR and DSC measurements to
study.FTIRabsorptionspectrawereanalyzedwithtwometh- eachotherandtherewithMCsimulationsprovedanothertime
ods.Onefollowedthetemperaturedependenceoftheabsorp- to bea usefulandpowerfultoolinexploringmeltingtransi-
tion maxima of the symmetric or asymmetric CH2 or CD2 tionsinlipidmembranes.
stretch vibrations. The other studied temperaturedependent
changesofthepeakareaofthedifferencespectra. Monitor- FTIR,DSCandMCSimulations
ing the positions of band maxima leads to incorrect results,
butthesimulateddatadescribewellthecurveshapesobtained FTIRallowedustomeasuretheabsorptionspectraoflipid
withtheDifferenceSpectramethod.Oursimulationsdescribe suspensionsconsistingofMLVswithvaryingDMPC-d54and
well the evolution of the area changes. Melting of single DSPC ratiosindependenceoftemperature. Inparticularwe
componentscannotbeinvestigatedinDSC.DSCaloneonly probedtheCH2andCD2vibrationalmodesofthelipidfatty
showsthebulkbehavior. InFTIRspectroscopyitispossible acidchains.Thedeuteriumisheavierthanthehydrogenatom
to separate the lipid species individually. Therefore, using which is why the symmetric and asymmetric bands of the
MC simulations we were able to link the two experimental CH2 andCD2 stretch vibrationsdisplayabsorptionsat dif-
MeltingofSingleLipidComponents 9
ferentwavenumbers. Thismadeitpossibletodistinguishthe well.
DMPC-d54andDSPClipids. Previously this model has been shown to be suited to de-
Weappliedtwodifferentmethodstoanalyzetheabsorption scribe heatcapacityprofilesas measuredwith DSC (54, 57,
spectrameasured. Onewasbasedonmonitoringthetemper- 59). Tenuniqueparametersareneededwhichwereobtained
aturedependenceofabsorptionmaximacorrespondingtothe from experimental heat capacity profiles. Six of them fol-
CH2andCD2stretchvibrations,respectively.Theotherone lowdirectlyfromtheheatcapacitycurvesoftheonecompo-
wasbuiltonfollowingchangesinthepeakareaofdifference nentsystems. Thesearetwiceentropyandenthalpychanges
spectra (Difference Spectra method). We comparedthe cor- andthecorrespondingcooperativityparameters.Themissing
respondingexperimentalresultstothetemperaturedependent fourparametershavetobefoundbyfittingtheheatcapacity
ratioofdisorderedchainsofthesinglelipidspeciesusingMC profiles of the binary mixtures. They must be and they are
simulationsofasimplemodel(seefig.3). thesameforallofthepossiblelipidratios. Duetotheerrors
in the c -profile measurements the parameter determination
ThesenumericalsimulationswerebasedonaDoniachmodel p
is subject to deviations from the ideal set of parameters. In
ofthelipidchainmeltingprocessassumingoneorderedand
our case we have taken the remainingfour parametersfrom
one disordered lipid chain state. Nearest neighbor interac-
the DMPC:DSPC system (59). This provedto be valid, but
tionsonlywereconsideredandthelipidchainswerearranged
small deviationsdueto this assumptioncannotbe excluded,
onatriangularlattice. Eventhoughitisaminimalisticmodel
too.
whichneglectsthatlipidchainsmayreflectdifferentdegrees
ofdisorderandthenon-considerationofalossoflatticeorder Heat capacity profiles contain macroscopic informationon
during the melting process the calculated fraction of disor- themeltingbehaviorsuchaschangesinenthalpyandentropy.
deredlipidchainsofthesinglelipidspeciesdescribestheex- However,DSC doesnotallowto determinethe microscopic
perimentaldataobtainedwiththeDifferenceSpectramethod behaviorofthemeltingprocess. Additionally,itdoesnotal-
low to investigate the melting of the single components. It
is only suited to measure the bulk melting behavior. FTIR,
however, providesa mean of studying the melting of single
lipid speciesandobservesthe microscopicdetails, butlacks
the possibility to probe macroscopic properties. The model
underlyingthe MC simulations describes heat capacity pro-
filesandpredictsthefindingsfromFTIRwell. Therefore,the
MCsimulationsprovideameantolinktheinformationofthe
twoexperimentaltechniques.
TheMeltingProcess
Themeltingprocessofsinglecomponentsisadequatelyde-
scribed using the numerical simulations. From the experi-
mentalandthesimulatedresultsandtheircorrespondingderiva-
tivesitbecomesclearthatthelowertemperaturemeltingcom-
Figure 5: Phase diagrams allow to obtain an under-
ponent (DMPC-d54) melts also in the binary mixture at a
standingofthephasebehavioroflipidmixturesindepen-
lower temperature than do the DSPC lipids (fig. 4). Still,
denceoflipidratios,temperature,pHorotherouterpa-
both lipid species melt over the whole temperature regime
rameters. Inthisstudybinarylipidmixtureswereusedto
andnotonlyatdistincttemperaturesatwhichfluctuationsas-
study melting transitions in dependence of temperature.
signedto thesinglelipid componentsareenhanced. Ingen-
Therefore, a phase diagram with the fraction of DSPC
eral,thecomponentwhichisintheminorityshowstwomax-
lipids as the abscissa and temperature as the ordinate
ima. Atoneofthesetemperaturesthestrengthofthefluctua-
was constructed. The open squares and the grey lines
tionsofthecomponentwhichisinthemajorityisthehighest.
represent the lower and upper temperature limits of the
This effect underlines the cooperativity of the melting pro-
meltingprocessasdeterminedfromheatcapacityprofiles
cess. Themeltingofonelipidincreasestheprobabilityofthe
simulatedusingthetangentmethod.Furtherthepeakpo-
meltingofanotherlipid.
sitionsofthederivativesofthedisorderedchainfractions
simulated(opencircles)andoftheexperimentallydeter- Thetemperaturesatwhichthefluctuationsofthetwocom-
mined area changes (crosses) were analyzed. The sim- ponentsisstrongestasobtainedfromthesimulationsandthe
ulatedpeaksdeterminedfrom thederivativesagreewith experiments, agree well (fig. 5). Further, the numerical
themaximaoftheheatcapacityprofiles(datanotshown) simulationsallowedtocalculatetheheatcapacityvaluesand
thedisorderedchainfractionssimultaneously. Therefore,we
wereabletocomparethetemperaturesatwhichtheheatca-
10 Fidorraetal.
pacity is in its maximum with the temperatures determined domains. Even if a membrane should be completely in the
fromthederivativesofthedisorderedchainfractions. These liquid disordered phase, lipids might not mix ideally and a
temperaturesagreewitheachother.Infig.1theheatcapacity heterogeneityexistsasseeninfig.1andasalreadydiscussed
profile of the equimolarmixture displays two maxima. The elsewhere(57). Thisstatementisalsotrueformembranesin
maximumat lowertemperaturecorrespondsto the enhance- the liquid ordered phase (74). The role of lipid membrane
mentin fluctuationsof the DMPC-d54 lipids and the one at heterogeneitieshas been discussed exhaustinglyin the liter-
highertemperaturestotheoneoftheDSPClipids. However, ature (25, 34, 38, 39, 40, 41, 43, 44, 45, 46, 47). Thereby,
thereareinstancesatwhichtheheatcapacityprofiledisplays ithasnotonlybeenpointedoutthatthecoexistenceofsolid
onlyonemaximumeventhoughonecandeterminetwotem- orderedandliquiddisorderedphasesmightbeimportant(39,
peratures using the evolution of the single lipid disordered 40,41,43,44),butalsotheenrichmentofsinglelipidspecies
chainratios. ThisisbecauseDSCdescribesthebulkbehav- incertainregionsofthelipidmembrane(42,45,46,47).
iorandsingleeventsmightnotbedetectablesincetheyshow Melting of single componentscannotbe detected from the
only a small contributionto the overallmelting. This needs bulk melting in the calorimeter. However, most of the pub-
attentionindiscussingmeltingprocessesinbiologicalmem- licationscitedherededucephaseseparationofnaturalmem-
branes. Inthecaseofbiologicalmembranesitmightevenbe branes from calorimetricresults. Therefore, details of these
difficulttodistinguishthemeltingprocessesfromthebaseline processes might not be revealed. It is also possible that the
ofthecalorimetricscan. Thismeansthatmeltingispresent, melting process in a biological membrane displays a broad
butitcannotbedetectedatallusingDSC. profileandtheheatchangesdetectedbyacalorimetercannot
bedistinguishedfromthebackgroundsignal.
FTIR:TheTwoApproaches In thiscontextnotonly the variouslipidmembranephases
have to be named, but also the possibility of enrichment of
In this paper we used two different techniques to analyze certainlipidspeciesin thecertainlipiddomains. Therefore,
theFTIRdata. Thesewerefollowingthetemperaturedepen- however,adetailedunderstandingoflipidmeltingtransitions
denceoftheabsorptionmaximaofeitherthesymmetricCH2 andlateralstructuringneedtobeobtained. Theexistenceof
ortheasymmetricCD2stretchvibrationsandtheareachange heterogeneitiesdependsonthethermodynamicalbehaviorof
ofoneofthepeaksofadifferencespectra. thedifferentlipidspecies.Asseeninthisstudytheymeltover
Usingthefirstmethodwefound,inaccordanceto(28),that a broad temperature regime with particular temperatures at
theabsorptionmaximumoftheCH2stretchvibrationsofthe whichtheydisplaystrongfluctuationsinmorecomplexlipid
DSPC lipidsdevelopedtowardssmaller wavenumberswhen mixtures. These temperatures depend on the lipid species,
the DMPC-d54 started to display stronger fluctuations (see but also on its interactionswith otherkindsof lipid present.
therightpanelsoffig.3). Ithasbeeninterpretedthatinthese Melting processes mightnot be finished even thougha heat
cases the DSPC lipids start to order. Concluding from the capacityprofilemightnotindicateanysuchevents. Thesen-
data presentedin this workand by Leidyet al. (28) this or- sitivitymightbenotsufficient. FTIRspectroscopy,however,
deringshouldbestrongerthaninthepresenceofacomplete provides means to distinguish the melting of single compo-
solidordered membrane. Thewavenumberatwhichtheab- nents.
sorptionisatamaximumdropstowavenumbersevensmaller Apossiblereasonforthebigvarietyinlipidspeciesmightbe
thanatlowtemperatures. Thereis, however,nofurthersup- to ensure a neededlipidmembraneheterogeneityandthere-
porting evidence that lipids might be able to order stronger withtoplayarolemembranefunction.Thisthenprovidesan
than at low temperatures. This behavior is in disaccord of explanationwhyadependenceoflipidsynthesizeinbiologi-
whatwe havefoundwith thesecondmethodandthe Monte calmembranesonouterphysicalparameterssuchaspressure
Carlo simulations. Therefore, it seems reasonable that the ortemperatureisrequired.
DifferenceSpectra methodyieldsresultswhich describethe
meltingprocesscorrectlyandmonitoringbandshiftsonlycan Acknowledgments
leadtoincorrectresults.
M.FidorrawasfundedbyBioNetandtheVillumKannRas-
mussenFoundation.
BiologicalRelevance
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