Influence of free fatty acids on lipid membrane-nisin interaction - Saitta et al. - 2020 - Unknown

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Influenceoffreefattyacidsonlipidmembrane-nisininteractionSaitta,F.etal.Langmuir,2020SupportingInformationInfluenceoffreefattyacidsonlipidmembrane-nisininteractionFrancescaSaittaa,PaoloMottaa,AlbertoBarbirolia,MarcoSignorellia,CarmeloLaRosac,AnnaJanaszewskab,BarbaraKlajnert-MaculewiczbandDimitriosFessasa,*aDipartimentodiScienzepergliAlimenti,laNutrizioneel'Ambiente,DeFENS,UniversitàdegliStudidiMilano,ViaCeloria2,20133,Milano,ItalybDepartmentofGeneralBiophysics,FacultyofBiologyandEnvironmentalProtection,UniversityofLodz,141/143PomorskaSt.,90-236Lodz,PolandcDipartimentodiScienzeChimiche,UniversitàdegliStudidiCatania,VialeAndreaDoria6,95125,Catania,Italy*Correspondingauthor.E-mailaddress:dimitrios.fessas@unimi.it.Tel.:+390250319219Numberofpages:7Numberoffigures:6Numberoftables:1TableOfContentsEXPERIMENTALSECTION...........................................................................................................S2Nisinpurification............................................................................................................................S2Thermalanalysismeasurements.....................................................................................................S3DynamicLightScattering..............................................................................................................S5RESULTSANDDISCUSSION........................................................................................................S6InfluenceofFFAschemicalstructureonlipidmembranesthermalstability................................S6S1

1Influenceoffreefattyacidsonlipidmembrane-nisininteractionSaitta,F.etal.Langmuir,2020EXPERIMENTALSECTIONNisinpurificationFigureS1showstheSDS-PAGEgelelectrophoresisdepictingthepurificationofnisin.Asrevealedbythefirstthreelanesatdifferentnisinconcentrations,thepurificationprotocolappliedtothecommercialpowderprovidedahighlypurifiedpeptide(nopeptidesotherthanthenisinbandatabout3.5kDaarevisibleinthegel).Sucharesultwassupportedbyboththelastthreelanes,whichwereperformedathighnisinconcentrationsinordertobetterdetectanypossiblecontaminant.HPLCchromatogramreportedinFigureS2quantifiedthepuritygradeofprotein,whichwas>95%asreportedintheexperimentalsection.FigureS1.SDS-PAGEgelelectrophoresisforthepurifiednisin.Thefirstthreelanesandthelastthreeonescorrespondtonisinsolutionsatdifferentconcentrations.3.02.52.01.51.0Absorbanceat215nm0.50.0-0.501020304050607080Elutiontime/minFigureS2.HPLCchromatogramforthepurifiednisin.S2

2Influenceoffreefattyacidsonlipidmembrane-nisininteractionSaitta,F.etal.Langmuir,2020ThermalanalysismeasurementsGenerally,theapplicationoftwoheating/coolingcyclestovesicledispersionsisenoughfortheachievementofequilibriumphases,allowingtheuseofthesecondheating/coolingcyclefortheanalysis.Inotherwords,suchcyclemaybeindicatedasthethermodynamicallymeaningfulonesinceanyotherfollowingcyclewouldleadtoalmostthesamecalorimetricprofile.However,lipidphasesinmetastableequilibriaandkineticphenomenamayariseifthephospholipidconstituentsdonotmanifestagoodthermodynamiccompatibility.Inthiscase,moreheating/coolingcyclesmightbenecessary.AsfarastheDMPC:DPPS3:2systemisconcerned,theapplicationoftheliposomepreparationprotocolallowedagoodmixingoftheconstituent,somuchtoobtainamicro-DSCthermogramwithawelldispersedandquitehomogenouscalorimetricprofileatthefirstheatingramp.However,thelipidphasesstartedtosegregatebecauseofthelowthermodynamiccompatibilityoftheconstituentsfromthenext(second)heatingscan.Themicro-DSCthermogramsderivingfromtheapplicationofthethirdandfourthheating/coolingcyclestothisbinarymembraneareshowninFigureS3.Weobservethatthefourthheatingscan(reportedasadashedblacktraceandcorrespondingtotheblackcurvereportedinFigure1ofthemaintext)isperfectlysuperimposabletothethirdheatingscan(reportedasasolidredtrace),revealingtheachievementofaphaseequilibrium.Thefourthheatingscanwasconsideredasthermodynamicallymeaningful,analogouslytowhatwasdoneforalltheothersystemsconsideredinthiswork.FigureS4reportsthecalorimetricprofilesobtainedforthemodelmembrane(5.7DMPC:3.8DPPS:0.5DOPCmolarratio)fortheapplicationofaheating/coolingcycleasanexample.Weobservereversiblegel-to-liquidcrystallinephasetransitionsasrevealedbytheexothermictraceduetothecoolingscan(dashedcurve).Asimilarbehaviorisobservedforallthesystemsconsideredinthiswork.S3

3Influenceoffreefattyacidsonlipidmembrane-nisininteractionSaitta,F.etal.Langmuir,20205ThirdheatingscanFourthheatingscan4endo1-3·mol1-2/kJ·KexcpC10-1152025303540455055T/°CFigureS3.Micro-DSCprofilesforDMPC:DPPS3:2vesiclesobtainedattheapplicationofthethird(solidredcurve)andfourth(dashedblackcurve)heatingramps.32endo1-1·mol1-0/kJ·KexcpC-1-2-351525354555T/°CFigureS4.Micro-DSCprofilesforvesiclesobtainedasa5.7DMPC:3.8DPPS:0.5DOPCmolarratio.Thethermogramscorrespondtothefourthheatingandcoolingscans(solidanddashedtraces,respectively).S4

4Influenceoffreefattyacidsonlipidmembrane-nisininteractionSaitta,F.etal.Langmuir,2020DynamicLightScatteringTableS1showsthedataobtainedfromtheDLSanalysisonvesiclescontaining20%ofFFAsandthathadalreadyundergonefourheating/coolingcyclesthroughmicro-DSCinordertoverifythattheintegrityofthevesicleswasnotcompromised.SuchscannedSUVsdispersionswereaddressedtothepreparationofnisin-containingsamples.Asshowninthetable,theliposomesizeresultedtobeaffectedbytheapplicationofmultipleheating/coolingcyclesdependingonthetypeofFFAincorporatedwithinthebilayer.Indeed,unlikethevesiclescontainingunsaturatedFFAswhichremainedunaffected,wehypothesisethatalimitedfractionoftheliposomescontainingsaturatedFFAsunderwentfusion,leadingtoaconsiderableincreaseofthePDIs(wecanexcludeaggregationorotherseveredamagesbecausetheywouldhavebeenvisibleandidentifiedonthemicro-DSCthermogramsastheywouldhaveproducedverydifferentprofilesthantheunilamellarvesicles).Nevertheless,therevealedmodificationsinvesiclesizeandPDIareunabletoproducemodificationsinthemicro-DSCthermograms,asreportedintheliteratureformulticomponentsystems(Saitta,F.etal.ColloidsandSurfacesB:Biointerfaces176(2019)167–175).TableS1.PhysicochemicalcharacteristicsobtainedfromDLSmeasurementsforseveralvesiclescontaining20%ofFFAs.Thereportedparametersarez-Averagediameter(Zave)withstandarddeviation(SD1)andthepolydispersityindex(PDI)withstandarddeviation(SD2).Thelabelsreportedonthetableindicatepalmiticacid(PA),stearicacid(SA),elaidicacid(EA),oleicacid(OA),linoleicacid(LA)anddocosahexaenoicacid(DHA).Z12aveSDPDISDnmnmModelmembrane(REF)84.20.60.2790.007REF+PA103.00.90.4440.010REF+SA126.91.20.5880.011REF+EA87.50.90.3250.002REF+OA67.40.20.1030.005REF+LA69.40.20.0740.004REF+DHA75.20.10.0700.028S5

5Influenceoffreefattyacidsonlipidmembrane-nisininteractionSaitta,F.etal.Langmuir,2020RESULTSANDDISCUSSIONInfluenceofFFAschemicalstructureonlipidmembranesthermalstabilityFigureS5shows,asanexample,themicro-DSCgel-to-liquidcrystallinephasetransitionobtainedforlinoleicacidandthesuperimpositionofthesigmoidaltrendoffluorescenceanisotropyvalues,r,againsttemperature.Weobservedthatthefalloftheanisotropyvalueswellmatchedthetemperatureregionofthephasetransitionsincetheprobe’sfluorescenceanisotropyreflectsthelevelsoforderandpackingofphospholipidacylchains.Moreover,thesigmoidflexpointwascomparabletothe?̅obtainedfromthecalorimetriccurve.Indeed,theflexpointshouldtheoreticallycorrespondtoa50%degreeofadvancementoftheprocess,thusindicatinganaveragetemperatureofthetransition.FigureS6reportsacomparisonbetweenthe?̅valuesfrommicro-DSCcurves(dottedbluebars)andtheflexpointofthesigmoidsfromfluorescenceanisotropy(linedredbars)obtainedforthemodelmembranealoneandcontainingthe20%ofvariousFFAs.Asalreadymentionedinthemaintext,theflexpointsobtainedfromspectroscopicexperimentswereinaccordancewiththe?̅valuesofthecalorimetricones,confirmingonceagainthetypeofeffectproducedbyFFAswithdifferentchemicalstructure.Theslightdifferencesbetweenthetwovaluesmaybeascribabletothefewspectroscopicexperimentalpointandtothelowersensitivityofthespectroscopictechniqueforthedetectionoflipidphasetransitionsthanthecalorimetricone,madealsoworsebytheexperimentalconditionssincetheheatingrampforfluorescencemeasurementswasobtainedbyapplyingdiscretefixedtemperaturestepsof3°Ceach.S6

6Influenceoffreefattyacidsonlipidmembrane-nisininteractionSaitta,F.etal.Langmuir,20200,42,5Modelmembrane0,35+220%LinoleicAcid0,31,51-0,25·mol1-r10,2/kJ·Kexcp0,5C0,15endo00,10,05-0,551525354555T/°CFigureS5.Exampleofsuperimpositionoffluorescenceanisotropy(r)ofDPHinmodelvesiclescontaining20%oflinoleicacid(redtrace)totherespectivemicro-DSCprofile(greentrace).Thedashedlinesindicatethe?̅forthecalorimetriccurve(greenline)andtheflexpointofthesigmoidobtainedfromfluorescenceanisotropy(redline).Probe:lipidmolarratiowas1:500.45,0T(Transitionaveragetemperature)?̅40,0Sigmoidflexpoint35,0REF30,0C25,0°T/20,015,010,05,00,0+DHA+LA+OASimplifiedModel+EA+PA+SAmembrane(REF)FigureS6.Histogramrepresentationshowingthe?̅valuesfrommicro-DSCcurves(dottedbluebars)andtheflexpointofthesigmoidsfromfluorescenceanisotropy(linedredbars)obtainedforthemodelmembranealoneandcontainingthe20%ofvariousFFAs.Thelabelsreportedonthehistogramindicatedocosahexaenoicacid(DHA),linoleicacid(LA),oleicacid(OA),elaidicacid(EA),palmiticacid(PA)andstearicacid(SA).S7