EFFECT of HEAD GROUP and CURVATURE ON BINDING of the ANTIMICROBIAL PEPTIDE TRITRPTICIN TO LIPID MEMBRANES

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ContentslistsavailableatSciVerseScienceDirect

Chemistry

and

Physics

of

Lipids

jo u r n al h om epa ge: w w w . e l s e v i e r . c o m / l o c a t e / c h e m p h y s l i p

EFFECT

OF

HEAD

GROUP

AND

CURVATURE

ON

BINDING

OF

THE

ANTIMICROBIAL

PEPTIDE

TRITRPTICIN

TO

LIPID

MEMBRANES

José

Carlos

Bozelli

Jr

a

_,

_Estela

_T.

_Sasahara

a

_,

_Marcelo

_R.S.

_Pinto

b

_,

_Clóvis

_R.

_Nakaie

b

_,

_Shirley

_Schreier

a,∗

a_Department_of_{Biochemistry,}_Institute_of_Chemistry,_University_of_São_Paulo,_São_Paulo,_SP,_C.P._26077,_05513-970,_Brazil b_Department_of_Biophysics,_Federal_University_of_São_Paulo,_São_Paulo,_SP,_04044-020,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received30September2011

Receivedinrevisedform7December2011 Accepted9December2011

Available online 22 December 2011

Keywords:

tritrpticin,antimicrobialpeptide,micelles, bilayers,spectroscopictechniques,toroidal pore

a

b

s

t

r

a

c

t

Inthisworkweexaminetheinteractionbetweenthe13-residuecationicantimicrobialpeptide(AMP) tritrpticin(VRRFPWWWPFLRR,TRP3)andmodelmembranesofvariablelipidcomposition.Theeffecton peptideconformationalpropertieswasinvestigatedbymeansofCD(circulardichroism)andfluorescence spectroscopies.Basedonthehypothesisthattheantibioticactsthroughamechanisminvolvingtoroidal poreformation,andtakingintoaccountthatmodelsoftoroidalporesimplytheformationofpositive curvature,weusedlargeunilamellarvesicles(LUV)tomimictheinitialstepofpeptide-lipidinteraction, whenthepeptidebindstothebilayermembrane,andmicellestomimicthetopologyoftheporeitself, sincetheseaggregatesdisplaypositivecurvature.Inordertomorefaithfullyassesstheroleofcurvature, micelleswerepreparedwithlysophospholipidscontaining(qualitativelyandquantitatively)headgroups identicaltothoseofbilayerphospholipids.CDandfluorescencespectrashowedthat,whileTRP3bindsto bilayersonlywhentheycarrynegativelychargedphospholipids,bindingtomicellesoccursirrespective ofsurfacecharge,indicatingthatelectrostaticinteractionsplayalesspredominantroleinthelattercase. Moreover,theconformationsacquiredbythepeptidewereindependentoflipidcompositioninboth bilayersandmicelles.However,theconformationsweredifferentinbilayersandinmicelles,suggesting thatcurvaturehasaninfluenceonthesecondarystructureacquiredbythepeptide.Fluorescencedata pointedtoaninterfaciallocationofTRP3inbothtypesofaggregates.Nevertheless,experimentswitha watersolublefluorescencequenchersuggestedthatthetryptophanresiduesaremoreaccessibletothe quencherinmicellesthaninbilayers.Thus,weproposethatbilayersandmicellescanbeusedasmodels forthetwostepsoftoroidalporeformation.

1. Introduction

Thewidespreaduse(andoftenmisuse)ofconventional antibi-oticsinhumanandanimalhealthcarehaveelevatedtheoccurrence ofantibioticresistanceinthelastdecades(Lohner,2009).Indeed, in2001theWorldHealthOrganization(WHO)classifiedantibiotic resistanceasaprioritydisease(WorldHealthOrganization,2001). Onestrategythathasbeenconsideredtosolvethisproblemisthe useofantimicrobialpeptides(AMPs)asanewclassofantibiotics.

AMPs areimportant constituentsof thefirst lineof defense of theinnate immune system(Brogden, 2005).Since the isola-tion/characterizationofcecropins,magainins,anddefensinsinthe 1980’s,1,800AMPshavebeenidentifiedindifferentorganisms, suchasfungi,bacteria,plants,invertebrates,andvertebrates. Gen-erally,themainfunctionofAMPsistokillordecreasethegrowthof apathogenicmicroorganism,howeverrecentstudiesshowedthat

∗_{Corresponding}_author._Institute_of_Chemistry, _University_of_São_Paulo,_C.P. 26077,CEP05513-970,Tel.:+551130912179;fax:+551130912179.

E-mailaddress:schreier@iq.usp.br(S.Schreier).

theycanalsomodulatetheinnateandadaptiveimmunesystems

(Yangetal.,1999;Jenssenetal.,2006).Whileconventional

antibi-oticsnormallydisableorkillbacteriaoveraperiodofdays,AMPs act withinminutes (Blondelleet al., 1999). Furthermore,AMPs presentawidespectrumofactivity,actingagainstGram-positive andGram-negativebacteria,protozoa,fungi,virus,andmammalian cells(Brogden,2005).

ItwasoriginallybelievedthatAMPsactatthemembranelevel bytheinsertionand/ordamage/permeabilizationofthe cytoplas-micmembraneofthemicroorganismcell(Lehreretal.,1989;Wade

etal.,1990;Shai,2002;Huangetal.,2004;LohnerandBlondelle,

2005).Thesepeptidesareusuallyshort,cationic,andacquirean amphipathicconformationupon interactionwiththecell mem-brane.Threemodelsareoftenconsideredtoexplainmembrane permeabilization(Brogden,2005;Jenssenetal.,2006;Bechinger,

2011): barrel-stave (Ehrenstein and Lecar, 1977), carpet (Pouny

etal.,1992),andtoroidalpore(Ludtkeetal.,1996).Morerecently,

ithasalsobeenproposedthatinsomecases,somecationic antimi-crobialpeptidesmightactbypromotingclusteringofnegatively chargedphospholipidsonthebacterialmembrane,inparticular, theabundantlipidphosphatidylglycerol(EpandandEpand,2009).

Open access under the Elsevier OA license.

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DiscussionsofthemechanismofactionofAMPshavealsobrought upconsiderationsabouttheroleoflipidcomposition(Leeetal.,

2004;Suetal.,2011)andaboutthepeptide:lipidratio(Leeetal.,

2004)onthismechanism.Inaddition,recentevidencesalsosuggest thatatleastsomeofthesecanactatintracellulartargets(Brogden, 2005).However,even inthiscase,anAMPwouldhavetocross thecellmembranetoreachthecytoplasm,whichimpliesaninitial interactionat themembrane level. Thus, the studyof peptide-membrane interactionsand the elucidationof theeffects these peptidesexertatthecellmembraneareimportanttounderstand theirbiologicalactivity.

Inthis workwefocus ontheAMPtritrpticin (VRRFPWWW-PFLRR,TRP3),a13-residuecationicpeptide,whichbelongstothe cathelicidin family. The highArg (30%) and Trp (23%) content, togetherwiththreeconsecutiveTrpresidues,makethispeptide unique(Chanetal.,2006;Sitaram,2006).TRP3hasbroad antimi-crobial activity against both Gram-positive and Gram-negative bacteria,andsomefungi(Lawyeretal.,1996;Cirionietal.,2006;

Ghisellietal.,2006),inadditiontohemolyticactivity.However,the

hemolyticeffectonlytakesplaceatconcentrationsmuchhigher thanthoserequiredforantimicrobialactivity(Yangetal.,2002). TheexistingstudiesshowthatTRP3actsatthemembranelevel, butitsdetailedmechanismofactionisnotknown.

NMRstudiesshowedthat,whileTRP3interconvertsbetween differentconformationsinsolution,uponbindingtoSDS(sodium dodecylsulfate) micelles it acquiresan amphipathic conforma-tionwithtwoadjacentturnsaroundPro5_and_Pro9_,_and_the_three

Trpclustered together.Thisconformationpositions acluster of hydrophobic residues at the micelle-water interface while the flexibleN-andC-terminicontainingtwo positivelychargedArg residueseacharelocatedontheothersideofthestructure, fac-ingtheaqueousphase(Schiblietal.,1999).Similarconformations werefoundforseveralTRP3analoguesboundto dodecylphospho-choline(DCP)micelles(Schiblietal.,2006;Andrushchenkoetal., 2006).Studieswithmodel membranesshowedthat TRP3binds preferentiallytonegativelychargedbilayers,and,toalesserextent, tozwitterionicbilayers.Italsopromotescomposition-dependent leakage from the inner compartment of lipid vesicles (Schibli et al., 2002).In addition we showed that the peptidedisplays ionchannel-likeactivityinplanarlipidbilayersandweproposed thatthechannelstructureisthatofatoroidalpore(Salayetal.,

2004).

AccordingtotheShai-Matsuzaki-Huang(SMH)model(Zasloff, 2002), initially the peptide binds to the membrane interface, acquiring an amphipathic conformation and aligning its long molecularaxisparallel tothemembrane surface. This localiza-tionpushesthepolarheadgroupsofthelipids,leadingtoalocal inductionofpositivecurvature(Matsuzaki,2001).Theunfavorable energyofmembranedeformationreachesacriticalvalue,leading toadynamicporeformedbyasupramolecularpeptide-lipid

com-plex(Matsuzakietal.,1996),oratoroidalpore(Ludtkeetal.,1996;

Yangetal.,2000)whosepolarsurfaceisformedbythepolarface ofamphipathicpeptidesandthepolarheadgroupsoflipids.

Becauseofthehighcomplexityofbiologicalmembranes,one usuallyresortstobiomimeticmodelsystemsinordertoinvestigate theeffects ofcomposition and physicalproperties ofthe phos-pholipidmatrixinthemembranes.Thesemodelsincludeplanar lipidbilayers,multilamellarandunilamellarvesicles,micelles,and Langmuirmonolayers(Schreieretal.,2000;JelinekandKolusheva,

2005; Seddon et al., 2009). The interaction between TRP3 and

lipidmodelmembraneshasbeeninvestigated(Nagpaletal.,1999;

Schiblietal.,1999;Nagpaletal.,2002;Schiblietal.,2002;Yang

etal., 2002; Yang etal., 2003; Salayet al., 2004;Schibli et al.,

2006;Andrushchenko etal., 2006;Andrushchenko etal., 2007;

Andrushchenkoetal.,2008;Nguyenetal.,2011).Theaimofthis

workistocontributetotheunderstandingofthemechanismof

action of TRP3at themolecularlevel. Toachieve this goal,we performedCD(circulardichroism)andfluorescencespectroscopic studiesofthepeptideinsolutionanduponinteractionwithmodel membranes–micellesandbilayervesicles-ofvariablelipid compo-sition.Inparticularwefocusontheeffectofmembranecurvature ontheconformationalbehaviorofthepeptide.Sinceithasbeen proposedthatthemechanismofactionofTRP3involvesformation ofa toroidalpore,which,likemicelles,presentspositive curva-ture,westudiedtheinteractionwithmicellesinordertomimic themoleculararrangementofpeptideandlipidsinthetoroidal porestructure.

2. MaterialsandMethods

2.1. Reagents

Acrylamideandsodiumdodecylsulfate(SDS)werefrom Bio-Rad,Hercules,CA.L-␣-tryptophanandcardiolipinofbovineheart (CL)werefromSigmaChemicalCo.,St.Louis,MO. 1-palmitoyl-2-hidroxy-sn-glycero-3-phosphocholine (LPC), 1-palmitoyl-2-hidr-oxy-sn-glycero-3-phosphoethanolamine (LPE), 1-palmitoyl-2-hi-droxy-sn-glycero-3-[phospho-rac-(1-glycerol)] (LPG), 1-palmi-toyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG), and phosphatidylethanolaminetransphosphatidylatedfromegg phos-phatidylcholine(ePE)werefromAvantiPolarLipids,Alabaster, AL.

2.2. Peptidesynthesis

TRP3wassynthesizedmanuallyaccordingtothestandardN␣

-tert-butyloxycarbonyl(Boc)protectinggroupstrategy(Merrifield,

1963;StewartandYoung,1984)usingp-toluenesulfonyl(TOS)and

formyl(For)Boc-aminoacidderivativesofArgand Trp, respec-tively. Thesynthesis wasperformed asdescribed byMalavolta

et al. (2002). Analytical HPLC (Waters), LC/MS

(electrospray-Micromass)-massspectrometry(theoreticalm/z=1902.28;found: 1902)andaminoacidanalysis(Biochrom20Plus,fromAmersham Biosciences)wereusedtocheckthehomogeneityofpurifiedTRP3.

2.3. Preparationofmodelmembranes

2.3.1. LargeUnilamellarVesicles(LUV)

Phospholipidsweredissolvedinchloroformandlipidfilmswith thedesiredmolarratios(purePOPC,POPC:ePE2:1,POPC:POPG2:1, POPC:ePE:POPG1:1:1,ePE:POPG2:1,andePE:POPG:CL67:23:10; thelattermimicsthepolarlipid compositionof E.coli B(ATCC 11303)membranes)wereprepared in glasstubes aftersolvent evaporationwithastreamofnitrogen;finaltracesofsolventwere removedinavacuumchamberforatleast2h.Thedriedlipidfilm wassuspendedbyvigorousvortexinginMilliQwater,pH7.19(±

0.02),atroomtemperature.Thelipidsuspensionswere submit-tedtofivecyclesoffreeze-thawing followedbythirty passages throughtwostacked100nmpolycarbonatefiltersusinganAvestin extruderatroomtemperature.Lipidphosphorouswasdetermined accordingtoRouseretal.(1970).

2.3.2. Micelles

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190 200 210 220 230 240 250 260 -16

-12 -8 -4 0 4 8

A

[

] x 10

-3 (deg.cm 2.dmol -1)

(nm)

TRP3 in the presence of H O2

POPC POPC:ePE 2:1

190 200 210 220 230 240 250 260

-16 -12 -8 -4 0 4 8

B

[

] x 10

-3 (deg.cm 2 .dmol -1 )

(nm)

[ePE:POPG] 2:1 ( M)

0 50 100 150 200 500

190 200 210 220 230 240 250 260

-16 -12 -8 -4 0 4 8

C

[

] x 10

-3 (deg.cm 2.dmol -1)

(nm)

TRP3 in the presence of POPC:POPG 2:1 POPC:ePE:POPG 1:1:1 ePE:POPG 2:1

E. coli

Figure1.Far-UVCDspectraof25.6±1.3␮MTRP3(A)inaqueoussolutionandinthepresenceof500␮MPOPCandPOPC:ePE2:1;(B)inthepresenceofincreasingePE:POPG 2:1concentration(␮M):0;50;100;150;200;500;(C)inthepresenceofLUVofvariablelipidcomposition,POPC:POPG2:1;POPC:ePE:POPG1:1:1;ePE:POPG2:1;ePE:POPG:CL 67:23:10(E.coli);lipid:peptideratio20:1.pH7.19±0.02.

2.4. Circulardichroism(CD)studies

2.4.1. Titrationwithmodelmembranes

CDspectrawereacquiredatroomtemperatureinaJascoJ-720 spectropolarimeter.Sampleswereplacedin1.00mmopticallength quartzcells.Thefinalspectraweretheaverageof6scans,after subtractingthespectrumobtainedunderthesameconditionsofa samplewithoutpeptide.Spectrawerescannedfrom190to260nm at50nm.s−1_using_a₂_nm_slit._The_initial_peptide_{concentration}_was

25.6±1.3␮M.Thelipidconcentrationvariedfrom0to500␮Mand from0to2.0mMinLUVandmicelles,respectively.

2.5. Fluorescencestudies

2.5.1. Titrationwithmodelmembranes

Fluorescencespectrawereacquiredatroomtemperatureina HitachiF-4500equipmentinterfacedwithacomputer.The excita-tionwavelengthwas280nmandtheemissionwasscannedfrom 305to450nm,at60nm.min−1_,_with_the_{photomultiplier}_at₇₀₀_V,

usingexcitationandemissionslitsof2.5nm.Spectratakenunder

thesameconditionswithoutpeptideweresubtractedfrom spec-traofthesamples,anddilutioneffectswerecorrected.Theinitial peptideconcentrationwas10.2±0.9␮Mforstudieswithmicelles and5.9±0.6␮MforstudieswithLUV.Thelipidconcentration var-iedfrom0to300␮Mandfrom0to2.0mMinLUVandmicelles, respectively.

2.5.2. Fluorescencequenchingbyacrylamide

TheintrinsicfluorescenceofTrpresiduesinTRP3wasquenched by the titration with acrylamide in the concentration range 0–200mM.Stern-Volmerconstants(Lakowicz,2006)were deter-minedbymeansofequation(1):

F0

F =1+KSV[Q] (1)

whereF0andFarethefluorescenceintensitiesintheabsence

andpresenceofvariableacrylamideconcentrations,respectively, KSVistheStern-Volmerconstant,and[Q]istheacrylamide

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3. Results

3.1. BindingofTRP3toLUV

Figure1Apresentsthefar-UVCDspectraofTRP3insolution

andin thepresence ofLUV composedof zwitterionic phospho-lipids(purePOPC and POPC:ePE 2:1). Thespectrum ofTRP3 in solutionismainlycharacterizedbythepresenceofanegativeband centeredat225nm.Thisbandisascribedtothecouplingofthe

1_B

b transitionof tryptophanside chainswithtransitions ofthe

peptidebackboneand/orwithtransitionsofotherspatiallyclose aromaticresidues(GrishinaandWoody,1994).Thepresenceofthis bandinthefar-UVregionisadominatingfeatureintheCD spec-trumofTRP3andprecludestheanalysisofthespectrainterms ofsecondarystructure.Forthisreason,theCDspectrawere ana-lyzedbycomparingthespectralfeaturesobservedunderdifferent conditions,namely,insolution,anduponbindingtobilayersor micelles.Basedonthenotionthatthepartitionofmembrane-active peptidesintomembrane interfacesisgenerally followedbythe acquisitionofsecondarystructure(WimleyandWhite,1996),the spectralchangesobserveduponinteractionofthepeptidewiththe modelmembraneswereinterpretedasbeingduetotheacquisition ofsecondarystructurebythepeptide.Moreover,thisapproachis reinforcedbytheNMRdeterminationofthesecondarystructure achievedbyTRP3andsomeofitsanaloguesuponbindingtoSDS andDPCmicelles.Suchkindofanalysishasalreadybeenperformed inCDstudiesofTRP3-modelmembraneinteraction(Schiblietal.,

1999;Yangetal.,2002;Andrushchenkoetal.2006).

Essentiallynospectralchangeswereobservedinthepresence ofLUVofPOPC orPOPC:ePE2:1(Fig.1A),suggestingthatTRP3 didnotinteractwithzwitterionicmembranes.Incontrast,upon titrationwithincreasingconcentrationsofLUVcontaininga nega-tivelychargedphospholipid(ePE:POPG2:1),spectralchangeswere observedinthefar-UVCDspectrumofTRP3:thenegativebandat 225nmbecamemoreintenseandtwopositivebandsappearedat

ca.200nmand210nm(Fig.1B).Basedontheconsiderationsabove, thesespectralchangesweretakenasanindicationofacquisition of secondary structure by TRP3, as a consequence of peptide-bilayerinteraction.Thisinteractionwasexaminedasafunction of membrane composition. In allcases, the negatively charged phospholipidPOPGwasincludedinviewofthehighlyfrequent presenceofthephosphoglycerolheadgroupinthemembranesof bacteria(Goldfine,1984;Lohner,2001;EpandandEpand,2009).

Figure1Cpresentsthefar-UVCDspectraofTRP3inthepresence

ofLUVof POPC:POPG2:1,POPC:ePE:POPG1:1:1, ePE:POPG2:1, andePE:POPG:CL67:23:10.Thelattersystemcorrespondstothe qualitativeandquantitativecompositionofpolarlipidsinthe mem-branesofE.coliB(ATCC11303).TheFigureshowsthatthemain spectralfeaturesinthespectraofTRP3aresimilar,suggestingthat thepeptideacquiressimilarconformationsuponbindingtothese bilayers.

Fluorescence spectraalsomonitored thebinding of TRP3to bilayersofvariablelipidcomposition.TheshiftoftheTrpresidues fromthepolaraqueousphasetothelesspolarmembranous envi-ronment caused an increase in peptide fluorescence intensity, as well as a decrease in the wavelength of maximum emis-sion(␭emmax).IncontrastwiththeCDresults, fluorescencedata

indicateda weakinteractionbetweenTRP3andpurePOPC and POPC:ePE2:1.However,undertheexperimentalconditionsused, saturationwasnotachieved,precludingfurtheranalysisofthese data.SimilartowhatwasfoundintheCDstudies,thefluorescence spectraprovidedclearevidencefortheinteractionbetweenTRP3 andbilayerscontainingnegativelychargedphospholipids.Forthis reason,onlytheresultsobtainedwiththesemembraneswere ana-lyzedfurther. ThebindingisothermsofTRP3toLUVcontaining negativelychargedphospholipids indicatethatthefluorescence

0 40 80 120 160 200 240 280 320

-0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

(I - I

0

)/I

0

[LUV] ( M)

POPC:POPG 2:1 POPC:ePE:POPG 1:1:1 ePE:POPG 2:1

Figure2.Increaseoffluorescenceintensity(I-I0/I0,seetext)asafunctionoflipid concentration,forPOPC:POPG2:1,POPC:ePE:POPG1:1:1,andePE:POPG2:1.pH 7.19±0.02.

intensityincreasedwithincreasinglipidconcentrationuntil sat-uration(Fig.2).Table1presentsthevalues of(I-I0/I0)x100at

saturatinglipidconcentrations,whereI0 andIrepresentthe

flu-orescenceintensitiesat␭emmax(inthemembrane)inthespectra

ofTRP3intheabsenceandinthepresenceofLUV.TheTablealso informsthevaluesof␭emmaxforL-␣-tryptophaninaqueous

solu-tionandforTRP3intheabsenceandpresenceofLUV.Thevalue of ␭emmax for L-␣-tryptophan is that reported in theliterature

(Lakowicz,2006);thesmallervalueforTRP3inaqueoussolution

suggeststhattheTrpresiduesinthepeptidechainexperiencean environmentontheaveragelessexposedtosolvent.Acriterionfor theextentofpenetrationofTrpresiduesintothemembraneisthe blueshiftintheemissionspectrumasaconsequenceofpartitioning inamorehydrophobicenvironment.Table1showsthatthevalues of␭emmaxdecreasedinthepresenceofmembranes.Furthermore,

Table1

Increaseinintensity,(I-I0)/I0×100,and␭emmaxinthefluorescencespectraofL-␣ -tryptophanandTRP3intheabsenceandpresenceofLUVandmicellesofvariable lipidcomposition.pH7.19±0.02.

[(I-I0)×100/I0](%)a ␭emmax(nm)

Insolution

L-␣-tryptophan - 350.0

TRP3 - 347.0

LUV

POPC:POPG2:1 59b _344.2

POPC:ePE:POPG1:1:1 48b _342.6

ePE:POPG2:1 26b _345.8

ePE:POPG:CL67:23:10(E.coli) -c _345.2

Micelles

LPC 53d _344.8

LPC:LPE2:1 28d _346.2

LPC:LPG2:1 41d _341.0

LPC:LPE:LPG1:1:1 28d _346.2

SDS -e _346.0

a_I

0andIrepresentthefluorescenceintensityat␭emmaxintheabsenceandinthe

presenceofmembrane,respectively.

b_Values_obtained_at₃₄₅_nm_in_the_presence_of₂₁₀_␮_M_lipid.

c_It_was_not_possible_to_evaluate_the_fluorescence_increase_due_to_light_scattering

effects.

d_Values_obtained_at₃₄₅_nm_in_the_presence_of₁₀₀₀_␮_M_lipid.

e_The_increase_in_fluorescence_was_not_evaluated_due_to_the_interaction_of_the_peptide

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Table2

Stern-Volmerconstants,KSV,M−1,foracrylamidequenchingofthefluorescenceof

L-␣-tryptophanandofTRP3intheabsenceandpresenceofLUVandmicellesof variablelipidcomposition.pH7.19±0.02.

KSV(M−1)

Insolution

L-␣-tryptophan 17.7

TRP3 10.4

LUV

POPC:POPG2:1 2.5

POPC:ePE:POPG1:1:1 2.1

ePE:POPG2:1 2.1

ePE:POPG:CL67:23:10(E.coli) 2.8 Micelle

LPC 3.9

LPC:LPE2:1 3.4

LPC:LPG2:1 3.4

LPC:LPE:LPG1:1:1 3.3

SDS 8.1

thesmallblueshiftsobservedsuggestthattheTrpresiduesofTRP3 arelocatedontheaverageclosetothebilayer-waterinterface.

Wealsoevaluatedtheaccessibilityoftryptophanresiduesto thewatersolublequencheracrylamide.Theextentofaccessibility wasmeasuredbytheStern-Volmerconstant(KSV),whichdecreases

withthedecreaseinaccessibility(Table2).ThevalueofKSVforfree

L-␣-tryptophaninaqueoussolutionwassimilartothatreported inliterature(Tallmadgeetal.,1989);thesmallervalueforTRP3in aqueoussolutionisinagreementwiththesmallervalueof␭emmax

(Table1)andindicatesthattheTrpresiduesinthepeptideareless

accessibletoacrylamidethanthefreeaminoacid.Inthepresenceof negativelychargedbilayers,thevaluesofKSVdecreaseevenfurther

(Table2), oncemoreevincingthebindingofthepeptidetothe

negativelychargedbilayers.

3.2. BindingofTRP3tomicelles

InordertoanalyzetheroleofcurvatureonTRP3-model mem-braneinteraction,weexaminedtheeffectofmicellesonpeptide conformationalproperties.Thecompositionoftheaggregateswas chosensothattheheadgroupcompositionwouldbequalitatively andquantitativelythesameasthatofsomeofthebilayersstudied; thus,micelleswerepreparedfromlysophospholipids.Inaddition, weusedSDStocomparewithdataalreadyreportedinthe liter-ature.Micellescontainingmorethan50mol%LPEcouldnotbe prepared,probablyduetothefactthatthislipidhasaKraft tem-peratureofapproximately60◦_C₍_Slater_et_al.,₁₉₈₉_).

Thefar-UV CDspectra ofTRP3 undergochanges upon titra-tionwithincreasingconcentrationsofLPCmicelles(Fig.3A).The intensityofthenegativebandaround225nmincreasedanda pos-itivebandappearedatca.198nmwithashoulderaround210nm. Thesespectralchanges, suggestedthattheinteractionwithLPC micelles induced acquisition ofconformation. Furthermore,the spectrasuggest that TRP3 acquiressimilar conformations upon bindingtomicellesofdifferentlipidcomposition(Figure3B).The resultsshowedthat,incontrasttowhatwasfoundinthecaseof bilayers,besidesbindingtonegativelychargedmicelles,TRP3also bindstozwitterionicmicelles.Inaddition,thespectrawere dif-ferentfromthoseobtainedinbilayers,asseeninFigure4.Finally, itshouldbenoticedthatthespectraofmicelle-boundTRP3were similartothatobtainedinthepresenceofSDSmicelles,whichwas similartothosereportedintheliterature(Schiblietal.,1999;Yang

etal.,2002).

Fluorescence spectraalsomonitoredtheinteraction ofTRP3 withmicelles.Theintensityincreasedandthewavelengthof max-imal emission decreased in the presence of micelles. Figure 5

presentsthebindingisothermsforthedifferentlipidcompositions.

Alsointhiscase,asaturationbehaviorwasobserved.Thevaluesof (I-I0/I0)x100atsaturationaswellasthoseof␭emmaxaregivenin

Table1.Inadditiontheblueshiftsresultingfrombindingwerealso

small,indicatingthatthetryptophanresiduesarelocatedcloseto themicelle-waterinterface.Acrylamidequenchingstudiesyielded KSVvalues(Table2)lowerthaninsolution,againreflecting

pep-tidebindingtothemicelles.Interestingly,theTrpresiduesseem tobemoreaccessibletothequencherinmicellesthanin bilay-ers.ThemuchhighervalueofKSVinthepresenceofSDSmicelles

whencomparedtothelysophospholipidmicellescouldbeduetoa greaterexposureofthepeptideonthemicellesurface,possiblydue toalargereffectofelectrostaticofpeptide-detergentinteractions, and/ortoahigherpartitioningofacrylamideinthesemicelles.

4. Discussion

TRP3hasbroadantimicrobialactivityagainstGram-positiveand Gram-negativebacteria,aswellassomefungi(Lawyeretal.,1996;

Cirionietal.,2006;Ghisellietal.,2006),inadditiontohemolytic

activity.However,thehemolyticeffectonlytakesplaceat concen-trationsmuchhigherthanthoserequiredforantimicrobialactivity (Yangetal.,2002).TheexistingstudiesshowthatTRP3actsatthe membranelevel,althoughthedetailedmechanismofactionisstill notfullyunderstood.

Anincreasingamountofexperimentaldatademonstratesthe preferentialinteractionofpositivelychargedAMPswithnegatively chargedphospholipids(Whiteetal.,1995;WenkandSeelig,1998,

Sitaram and Nagaraj, 1999; Zasloff, 2002; Jenssen et al., 2006;

Lohner,2009).Thispreferencewouldberesponsibleforthe

selec-tivityofAMPtowardsmicroorganisms,inviewofthedifferences betweenthelipidcompositionofmammalianandbacterial mem-branes. Themainphospholipids intheouterleafletof bacterial membranesarePE(zwitterionic),PG,andcardiolipin(both neg-ativelycharged)(Goldfine,1984;Lohner,2001;EpandandEpand, 2009);thelattertwoimpartnegativechargeonthemembrane sur-face.Incontrast,themajorlipidsintheouterleafletofmammalian membranesarezwitterionicPCandsphingomyelin, andneutral cholesterol,whichleadstoanetsurfacechargeofapproximately

zero(RothmanandLeonard,1977).

Fluorescencestudies(␭emmaxblueshifts,red-edgeeffects,

fluo-rescencequenchingbywatersolubleand membraneembedded quenchers)showedthat TRP3bindspreferentiallytonegatively chargedmembranesand,toalesserextent,tozwitterionic mem-branes(Schiblietal.,2002).Isothermaltitrationcalorimetry(ITC) studiescorroboratedthesefindings(Andrushchenkoetal.,2008), pointingtotheroleofelectrostaticinteractionsforpeptide bind-ing.Withregardtotheeffectonbilayerpermeability,Schiblietal.

(2002)demonstratedthat TRP3inducesleakageofthecontents

fromtheinnercompartment oflipidvesicles.In addition,Salay

etal.(2004)showedthechannel-likeactivityofTRP3in planar

lipidmembranes(BLM)andSchiblietal.(2006)providedevidence forTRP3-inducedlipidflip-flop.Thesedatahavebeentakenas evi-dencesinfavorofatoroidalporemechanismforTRP3.

Itiswidelyacknowledgedthatthepartitioningofmembrane active peptides into membranes is generally followed by the acquisition of secondary structure (Wimley and White, 1996). NMR studies showed that, in the presence of anionic SDS and zwitterionicDPCmicelles,TRP3acquiresanamphipathic confor-mationwithtwo adjacentturnsaroundPro5 _and _Pro9_,_and _the

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260 250 240 230 220 210 200 190 -15 -10 -5 0 5 10 15

A

[

θ

] x 10

-3 (deg.cm 2.dmol -1)

λ

(nm)

[LPC] (µM) 0 50

150

500 1000

2000

260 250 240 230 220 210 200 190 -15 -10 -5 0 5 10 15

B

[

θ

] x 10

-3 (deg.cm 2.dmol -1)

λ

(nm)

TRP3 in the presence of

LPC LPC:LPE 2:1

LPC:LPG 2:1

LPC:LPE:LPG 1:1:1 SDS

Figure3.Far-UVCDspectraof25.6±1.3␮MTRP3(A)inthepresenceofincreasingLPCconcentration(␮M):0;50;150;500;1000;2000;(B)inthepresenceofmicellesof variablelipidcomposition:LPC;LPC:LPE2:1;LPC:LPG2:1;LPC:LPE:LPG1:1:1;SDS.Lipid:peptideratio60:1,exceptSDSwheretheratiowas1000:1.pH7.19±0.02.

membrane-waterinterface(WimleyandWhite,1996;Yauetal.,

1998).

DiscussionsconcerningthemechanismofactionofAMP,in par-ticularwhenthemechanismunderconsiderationistheformation oftoroidal pores,have increasingly focusedonthe role of cur-vature,morespecificallypositivecurvature,sincethemodelsfor toroidalporeformationimplythattheparticipatinglipidsacquire positivecurvature.Inthiscontext,oneshouldtakeintoaccount boththepropensityoflipidstoacquirepositivecurvatureandthe propensityofpeptidestoinducepositivecurvature.Sinceithas beenproposedthatTRP3actsbyatoroidalporemechanism(Vogel

etal.,2002;Schiblietal.,2002;Salayetal.,2004),themaingoalin

thisworkwastoinvestigatetheeffectofcurvatureonthe interac-tionbetweenTRP3andmodellipidmembranes.Forthispurpose, weexaminedtheconformationalpropertiesoftheTRP3with bilay-ers(LUV)andmicellesofvariablelipidcompositionbymeansof

CDandfluorescencespectroscopies.Thereasoningunderlyingthe choiceofmicelleswasthattheseaggregatesdisplaypositive curva-ture,therefore,thesestructureswould,tosomeextent,mimicthe molecularorganizationoflipidsandpeptidesinthetoroidalpore. Inordertocomparethedataobtainedforbilayersandmicelles, thelatterwerepreparedwithlysophospholipidscarryingthesame headgroupsasthoseofphospholipidsinLUV,atthesamemolar proportions.Thus,theonlydifferencebetweenthetwotypesof aggregateswasthemolecularorganizationofthecomponents.

Thepresenceofthreetryptophanresiduesposedaproblemwith respecttotheuseofCDtoobtaininformationaboutpeptide sec-ondarystructure.Theseresiduesgaverisetoa bandascribedto thecouplingofthe1_B

btransitionoftheTrpsidechainswith

tran-sitionsofthepeptidebackboneand/orwithtransitionsofother aromaticresiduessidechainsspatiallyclose(GrishinaandWoody, 1994)whichprecludedanalysisofthefar-UVCDspectrainterms

260 250 240 230 220 210 200 190 -12

-8 -4 0 4 8 12

[

θ

] x 10

-3 (deg.cm 2.dmol -1)

A

λ (nm)

TRP3 in the presence of POPC:POPG 2:1

LPC:LPG 2:1

260 250 240 230 220 210 200 190 -12

-8 -4 0 4 8 12

[

θ

] x 10

-3 (deg.cm 2.dmol -1)

B

λ (nm)

TRP3 in the presence of

POPC:ePE:POPG 1:1:1 LPC:LPE:LPG 1:1:1

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0 200 400 600 800 100012001400160018002000 0.0

0.2 0.4 0.6

A

(I - I

0

)/I0

LPC

LPC:LPE 2:1 LPC:LPG 2:1 LPC:LPE:LPG 1:1:1

0 20 40 60 80 100 120 140 160 180 200 0.0

0.1 0.2 0.3 0.4

B

(I - I

0

)/I0

[Micelles] ( M) [Micelles] ( M)

LPC LPC:LPE 2:1 LPC:LPG 2:1 LPC:LPE:LPG 1:1:1

Figure5.Increaseoffluorescenceintensity(I-I0/I0,seetext)asafunctionoflipidconcentration.(A)LPC,LPC:LPE2:1,LPC:LPG2:1,andLPC:LPE:LPG1:1:1.pH7.19±0.02.In (B)theconcentrationaxiswasexpandedtoallowthecomparisonbetweendataformicellesandbilayers.

ofsecondarystructure.Nevertheless,uponbindingofTRP3tothe modelmembranes,spectralchangeswereobserved,suggestingthe acquisitionofconformation.

Thus, it was possibleto observe differences between bilay-ersandmicelles:whileTRP3interactedwiththeformermostly whenthemembranescontainednegativelychargedphospholipids (Fig.1),revealingtheimportanceofelectrostaticinteractionsfor peptidebinding,thepeptidewasabletointeractwithboth zwitte-rionic(LPC,LPC:LPE2:1)andnegativelychargedmicelles(Fig.3). Similarresultswerefoundinotherstudies,wheretheinteraction ofpeptideswithmicelleswasseentobeindependentofthecharge atthepolarheadgroup(Pertinhezetal.,1995;Pertinhezetal.,

1997;Salinasetal.,2002).Suchfindingssuggestthatother

interac-tions(hydrophobic,hydrogenbonding,vanderWaalsinteractions, hydrationforces)haveagreatercontributiontotheenergeticsof bindinginmicelles.Severalstudiespointtotheformationof hydro-genbondsbetweentheguanidogroupofArgresiduesinpeptides andphospholipidphosphategroups(EpandandVogel,1999;Tang

etal.,2007;TangandHong,2009).Ithasbeenproposedthatthese

hydrogenbondsarethedrivingforceforporeformation.Recently, Nguyen and coworkers (2011)demonstrated theoccurrence of suchhydrogenbondsuponinteractionofTRP3withmodel mem-branes.

TheCDdataalsosuggestedthattheconformationofTRP3is sim-ilarinbilayers,irrespectiveoflipidcomposition.Thesameholds formicelles.Moreover,thesimilarityofthespectrain lysophos-pholipidmicellesandinSDSsuggeststhattheconformationofthe peptideintheseaggregatesisthatfoundbyNMRinSDSandDPC micelles(Schibliet al.,1999;Schibli etal.,2006).However,the conformationsaredifferentin micellesandbilayers,evenwhen bothshareidenticalheadgroups(Fig.4).Theseresultsstrongly suggest that the different conformations acquired by TRP3 are predominantlymodulatedbymembrane curvatureandthatthe conformationinmicellescouldberelatedtothatdisplayedbythe peptideintoroidal pores.Moreover,thedata alsoillustratethe mutualinfluenceoflipidandpeptidewithregardtopore forma-tion:whilethepeptidepromotestheacquisitionofcurvature,the lipidorganizationmodulatespeptideconformation.

The fluorescence data corroborate the CD results in show-ing that, while TRP3binds tobilayers only when theycontain

negativelychargedphospholipids(Fig.2andTable1),the inter-actionwithmicellesisindependentofsurfacecharge(Fig.5and

Table1).Theresultsalsopointtothelocationof TRP3closeto

themembrane-surfaceinterfaceinbothbilayersandmicelles,as evincedbythesmallblueshiftsinthevaluesof␭emmaxwithrespect

tothat inwater (Table1).Theseresultsarein agreementwith previousobservations(Schiblietal.,1999;Schiblietal.,2002). Nev-ertheless,thehighervaluesoftheStern-Volmerconstants(Table2) suggestthatthelocationofTRP3inmicellesismoresuperficial.This conclusion,however,shouldbetakenwithsomecaution,sinceit shouldbeconsideredthatthequenchermighthaveagreaterdegree of partitioninginto micelles,as discussedby Eftinkand Ghiron

(1976)inthecaseofSDS.

Sincetheexperimentalapproachesusedinthisstudyfocusedon thepeptide,itwasnotpossibletoexaminethequestionofwhether thepeptide-membraneinteractionpromotedlipidclustering;this questionwillbedealtwithinfuturework.

Possibleimplicationsofthepresentdataforthemechanismof

toroidalporeformationbyTRP3

Theresultsobtainedinthisworkprovidesubsidiesforthe elu-cidationofthemechanismofporeformationbyTRP3.Salayand coworkers(2004)demonstrated theion channel-likeactivityof TRP3andproposedthatthemechanismofactionofthepeptide involvestheformationofatoroidalpore.Thefollowingsequence ofeventsisthoughttoleadtoporeformation:initially,thepeptide bindsparalleltothemembranesurfaceatthemembrane-water interface; subsequently, accumulatedpeptide molecules induce formationoflocalpositivecurvaturewiththeparticipationoflipids thathaveapropensitytoformpositivecurvature(e.g.,PGandPC,

LohnerandBlondelle,2005).ThecartooninFigure6depictsthis

sequenceofevents.

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Figure6.Cartoondepicting(a)agenericpeptideinsolution,(b)peptideboundto thebilayersurface,(c)toroidalporeshowingthemicelle-likelipidorganization. Between(b)and(c),ontheright,topviewofthetoroidalpore.Reproducedfrom Toke,2005,withpermissionofJohnWiley&Sons.Ontheleft,theSDS-boundribbon conformationofTRP3(pdb1d6x),asdeterminedbyNMRbySchiblietal.,1999.

theinitialstepofTRP3bindingtothemembrane,whiletheresults formicellesarerelatedtothegeometryofbothlipidandpeptide moleculesinthepore.Asaconsequence,wesuggestthatmicelles canbeusedtomimicthemoleculararrangementoflipidsand pep-tidesinthetoroidalpore.

Acknowledgements

Thisresearchwassupportedbygrants fromFAPESP.J.C.B.Jr. hadCNPqM.Sc.fellowshipandpresentlyhasaPh.D.fellowship, M.R.S.P.isaM.Sc.student,E.T.S.hadaPIBICundergraduatestudent fellowship,C.R.N.andS.S.areCNPqresearchfellows.

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