Conformational and thermal characterization of a synthetic peptidic fragment inspired from human tropoelastin: Signature of the amyloid fibers

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OULOUSE

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Dandurand, Jany and Samouillan, Valérie and Lacoste-Ferré,

Marie-Hélène and Lacabanne, Colette and Bochicchio, Brigida and Pepe,

Antonietta Conformational and thermal characterization of a

synthetic peptidic fragment inspired from human tropoelastin:

Signature of the amyloid fibers. (2014) Pathologie Biologie, vol. 62

(n° 2). pp. 100-107. ISSN 0369-8114

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Conformational

and

thermal

characterization

of

a

synthetic

peptidic

fragment

inspired

from

human

tropoelastin:

Signature

of

the

amyloid

fibers

Caracte´risation

conformationnelle

et

thermique

d’un

fragment

peptidique

synthe´tique

inspire´ de

la

tropole´lastine

humaine

:

signature

des

fibres

amyloı¨des

J.

Dandurand

a

,

V.

Samouillan

a,

*

,

M.H.

Lacoste-Ferre

a

,

C.

Lacabanne

a

,

B.Bochicchio

b

,

A.

Pepe

b

a_{PhysiquedesPolyme`res,CIRIMATUMR5085,InstitutCarnot,Universite´ PaulSabatier,118,routedeNarbonne,31062Toulousecedex,France} b_{DepartmentofSciences,UniversityofBasilicata,ViaAteneoLucano,10,85100Potenza,Italy}

Keywords: Elastin Syntheticpolypeptide Amyloidfibers Thermalanalysis Secondaryconformation Motscle´s: E´lastine Peptidesynthe´tique Fibresamyloı¨des Analysethermique Conformationsecondaire ABSTRACT

Objectives.–This work dealswiththe conformationaland thermal characterizationof asynthetic

peptide(S4)releasedduringtheproteolysisofhumantropoelastinbythematrixmetalloproteinase-12

thatwasshowntoformamyloid-likefibresundercertainconditions.

Materialsandmethods.–S4peptidesweresynthesizedbysolid-phasemethodologyandaggregatedin

solutionat808C.Fouriertransform–infraredspectroscopy(FT–IR)wasusedtoaccessthesecondary

structure. Thermal characterization was performed by thermogravimetric analysis (TGA) and

differentialscanningcalorimetry(DSC).

Results.–TheDSCstudyofthesolublelinearpeptideS4insolutioninTBSrevealstheirreversible

aggregationintoamyloid fibres.FT–IR,DSCand TGAanalyses performedonfreeze-dried samples

evidencedifferencesbetweenthelinearpeptideanditsassociatedamyloid-likefibres, bothonthe

conformationandthephysicalstructure.WhenS4peptidesareaggregated,theprominentconformation

scannedbyFT–IRisthecrossb-structure,correspondingtoTGAtoanincreaseofthethermalstability.

Moreover,theDSCthermogramsofS4fibresarecharacteristicofahighlyorderedstructure,incontrast

totheDSCthermogramsofS4linearpeptides,characteristicofanamorphousstructure.Finally,theDSC

analysisofdifferentlyhydratedS4 fibresbringsto thefore thespecificthermalanswerofthewet

interfacesofthecrossb-fibres.

Conclusion.–FT–IRandthermaltechniquesarewellsuitedtoevidenceconformationalandstructural

differencesbetweenthesolublepeptideanditsamyloidform.

RE´ SUME´

Butdel’e´tude.– Cettee´tudeconcernelacaracte´risationphysiqueetconformationnelled’unpeptide

synthe´tique(S4)libe´re´ lorsdelaprote´olysedelatropoe´lastinehumaineparlame´talloprote´ase-12etqui

peutformerdesfibresamyloı¨des souscertainesconditions.

Mate´riauxetme´thodes.–LespeptidesS4sontsynthe´tise´senphasesolideetagre´ge´sensolutiona` 808C.

Laspectroscopieinfrarougea` transforme´edeFourier(IR–TF)estutilise´epouracce´dera` lastructure

secondaire.Lacaracte´risationthermiqueesteffectue´eparanalysethermogravime´trique(ATG)etpar

analysecalorime´triquediatherme(ACD).

Re´sultats.–L’analyseparACDdupeptidesolubleetline´aireensolutiondansleTBSre´ve`lel’agre´gation

irre´versibleenfibresamyloı¨des. LesanalysesparIR–TF,ACDetATGsurlese´chantillonslyophilise´s

* Correspondingauthor.

E-mailaddress:valerie.samouillan@univ-tlse3.fr(V.Samouillan). http://dx.doi.org/10.1016/j.patbio.2014.02.001

1. Introduction

Elastin,theproteinresponsibleforelasticityoftissues,suchas lung, skin and arterial walls consists of a three-dimensional network whose turnover is almost absent under physiological conditions.However,underpathologicalconditions,theelastolysis bymatrixmetalloproteinase-12(MMP-12)canproducebiological active fragments as it was shown in photo-damaged skin [1], arteries of atherosclerotic patients [2] and in lung where the elastoticmaterialcontainsamyloid-likefibres[3].

Moreover,anex-vivostudyevidencedamyloid-likefibresonly reacting with a polyclonal antibody to human elastin within atherosclerosisplaquesofcerebralarteriesofsenilepatients[4], suggesting the involvement of elastin in the amyloidogenic process.Interestingly,theextensiveinvestigationbymass spectro-metryofthecleavagesitesofMMP-12inhumanskinelastin[5]

showed that polypeptide sequences encoded by exon 30 were presentintheelastinlysate.Giventhesefinding,wehypothesize that elastin-derived soluble peptides released from elastin by elastolysismightaggregateintoamyloidstructuresinthevicinity of thedegraded elastinfibres,thatcouldexplainthe‘‘elastotic’’ materialdescribedinnumerousreports.

Forthefirsttime,somepolypeptidesequencesencodedfrom exon30(theentirepolypeptidicsequenceencodedbyexon30) waspreviouslydemonstratedtobeamyloidogenic[6],mimicking theelastinfragmentsproducedbyelastasesweresynthesizedand studiedbothatthemolecularandsupramolecularlevelsbyvarious spectroscopies,MDsimulationsandAFMmicroscopy[7].Among thesepeptides,thelongestone(S4peptide)wasshowntogiverise toamyloidfibres.

The aim of this workis to get an insight into the thermal propertiesofthesoluble,linearpeptideS4(labelledS4peptides) anditsaggregatedform(labelledS4fibres)mainlyusingthermal analysis (TGAand DSC). DSCis a well-suitedtechnique for the analysisofbiologicalmaterialsbothinsolution[8–10]andinthe condensedstate[11–14],theinsolubilityofsomefibresbeingnot an obstacletotheanalysis.Combined withTGA,DSCallows to distinguishandtoestimatetheamountofthedifferenttypesof waterinproteins.Moreover,thesetechniquesrevealorderedand amorphous structures at the mesoscopic level, connecting the molecular level (accessible by vibrational techniques) and the microscopic/macroscopic level (accessible by microscopy and

turbidimetry),whichwerealreadyexploredinthepreviouswork onS4[7].InTable1,wesummarizethedifferenttechniquesused inthisstudyfocusingonthespecificcontributionofeachofthem. Ithaslongbeenrecognizedthatamyloidfibrilscontaina

cross-b

spine,with

b

-strands perpendiculartothefibrilaxis[15,16]. Atomicstructures havebeendeterminedby X-rayanalysis[17]

andbymoleculardynamicssimulations[18–20]forsomeofthese cross-

b

spines,revealingapairof

b

-sheetsmatedcloselytogether by intermeshing side chainsin what hasbeen termeda steric zipper[17].Thefirstlevelintheaggregationofamyloidstructures istherapidandreversibleformationof

b

-sheets[16].Theslower secondstep,accompaniedbythedecreaseofentropy,consistsin thedehydrationandinterdigitationofthematingsheet,allowing theformationofthedryamide-stackinghydrogenbonds.

Froma medicalpointofview, thermodynamicand solvation structural properties of amyloidogenic polypeptide sequences couldhelpourunderstanding onthemechanism ofaggregation andtheveryhighinsolubilityofamyloidfibresalsoinvolvedin neurodegenerativediseases[21,22].Fromamaterialpointofview, theexcellentthermalstabilityandenhancedmechanical proper-tiesofamyloidfibresmakethesestructurespromisingcandidates for biomaterial development [23], and their structure–function relationshipdeservestobeclarified.

2. Materialsandmethods 2.1.Peptidesynthesisandpurification

TheS4peptides(LVGAAGLGGLGVGGLGVPGVGG,molecularweight:1734g/mol) weresynthesized bysolid-phasemethodologyusinganautomatic synthesizer APPLIEDBYOSYSTEMmodel431A.Fmoc/DCC/HOBTchemistrywasused,starting from(0.25mM)Wangresin(NovaBiochem,Laufelfingen,Switzerland).The Fmoc-aminoacidswerepurchasedfromNovaBiochemandfromInbios(Pozzuoli,Italy). Thecleavageofthepeptidesfromtheresinwasachievedbyusinganaqueous mixtureof95%trifluoroaceticacid.TheS4peptideswerefreeze-driedandpurified byreversed-phasehighperformanceliquidchromatographyandtheirpuritywas assessedbyelectrospraymassspectrometry.

2.2.FormationofS4fibres

Accordingtopreviousturbidimetryexperimentsshowingtheself-aggregationof S4intoamyloid-likestructures[7],S4peptidesweredissolvedinTBSbuffer[Tris (50mM),NaCl(1.5M),andCaCl2(1.0mM),pH7]withaconcentrationof30mg/mL

(17mM);thesolutionwasstirredat808Cfor3hinordertoprecipitateS4peptides intoS4fibres.Onceformed,S4fibreswereseparatedfromthesolutionby3cyclesof centrifugation(15,000rpm,5min)andwashedinpuredistilledwaterpriorthe freeze-dryingprocess.

2.3.Fouriertransform–infraredspectroscopy(FT–IR)

Fouriertransform–infraredspectroscopy/attenuated totalreflectance(FT–IR/ ATR)spectrawerecollectedusingaNicolet5700FTIR(THERMOFISHERSCIENTIFIC, Waltham,MA)equippedinATRdeviceequippedwithaKBrbeamsplitteranda MCT/Bdetector.Spectrawererecordedovertheregionof4000–450cm1_with

aspectralresolutionof4cm1

and64accumulations.TheATRaccessoryusedwasa SmartOrbitequippedwithatypeIIAdiamondcrystal(refractiveindex2.4).A single-beambackgroundspectrumwascollectedfromthecleandiamondcrystal beforeeachexperimentandthisbackgroundwassubtractedfromthespectra.

mettentene´videncedesdiffe´rencesentrelaformesolubleetlaformeamyloı¨de, tantauniveaudes

conformations quede lastructure physique.Quand lespeptides S4 sontagre´ge´s, laconformation

pre´ponde´rantere´ve´le´eparIR–TFestlastructureenfeuilletsbcroise´s,correspondantenATGa` une

augmentation de la stabilite´ thermique. De plus, les thermogrammes ACD des fibres S4 sont

caracte´ristiquesd’unestructurehautementordonne´e,contrairementauxthermogrammesdespeptides

S4,associe´sa` unestructureamorphe.Enfin,l’analyseACDdesfibresS4diffe´remmenthydrate´esmeten

e´videncelare´ponsethermiquedel’interfacehumidedesfibresenfeuilletsbcroise´s.

Conclusion. –Lestechniquesvibrationnellesetthermiquessontbienadapte´espourmettreene´vidence

lesdiffe´rencesconformationnellesetstructuralesentrelepeptidesolubleetsaformeamyloı¨de.

Table1

Specificcontributionsofthetechniquesusedinthisstudy.

Techniques Contributions

Fouriertransform–nfra-redspectroscopy(FT–IR) Secondarystructures Thermogravimetricanalysis

(TGA)

Hydrationandthermal stability

Watersorption Wateraffinity

DifferentialScanningCalorimetry(DSC) Orderedanddisordered structures

Fourier-self-deconvolution(FSD)oftheinfraredspectrathatallowsresolutionof severaloverlappingbands[24]wasperformedintheamideI/IIregionusingOmnic software(THERMOFISHERSCIENTIFIC,Waltham,MA)toextractthepeakmaxima. ThedecompositionoftheamideI/IIbandsontherenormalizedspectrumwasthen performedwithaGaussiancurvefittingprocedure.

2.4.Thermogravimetryanalysis(TGA)

Analyseswereperformedintriplicateonfreeze-driedsamples(initialweight 5mg) set in alumina pans between 25 and 6508C at 108C/min under N2

atmosphereusingaTGAQ50(TAINSTRUMENTS,NewCastle,DE). 2.5.Watersorption

Initial freeze-dried samples were equilibrated on MgN2O66H2O saturated

solutions at258C (correspondingto a 53%relativehumidity). Samples were weightedwithaMettlermicrobalanceafterdifferenttimesofequilibrium.After measurementssampleswerecompletelydehydratedoverP2O5overnightat1058C

andweightedagaintodeterminethemassofthedrysamplesandthemassofwater bydifference.Thehydrationlevelwasdefinedasthemassofwaterdividedbythe massofthehydratedsample.

2.6.Differentialscanningcalorimetry(DSC)

Analyses were performed using a DSC Pyris calorimeter (PERKIN ELMER, Waltham,MA).Thecalorimeterwascalibratedusingcyclohexaneandindiumas standards,resultinginatemperatureaccuracyof0.18Candanenthalpyaccuracy of0.2J/g.Thesamplecellwaspurgedwithheliumandthecoolingandheatingscan rateswere10or208C/mindependingontheexperiments.

Anamountof10mLofS4peptidesolutions[2mMinsolutioninTBSbufferTris (50mM),NaCl(1.5M),andCaCl2(1.0mM),pH7]weresealedin15mLaluminium

hermeticpans.Experiments,doneintriplicate,wereperformedbetween10and 908Cintheheatingandcoolingmodeat108C/min.

Freeze-driedS4peptidesandfreeze-driedS4fibres,5mginweight,weresealed into non-hermetic aluminium pans. Experiments, done in triplicate, were performedbetween5and2258Cwithaheatingrateof208C/min.

HydratedS4fibers,2–10mginweight,weresealedintohermeticaluminium pans.Sampleswerefirstcooledat–508Cat108C/minandequilibratedat–508C during5min.Experiments,doneintriplicate,wererecordedintheheatingmode between–50and30–708C(dependingonthehydrationlevel)withaheatingrateof 208C/min.

3. Results

3.1. DSCanalysisofS4peptidesinsolution

During the first heating, the thermogram of S4 peptides in solution(Fig.1)ischaracterizedbythe

D

Cpheatcapacitystepat

378C and two sharp exothermic peaks at 618C and 788C, superimposed on

D

Cp heat capacity steps; in contrast, the

thermogram recorded during cooling does not present any transitioninthe80–608Ctemperaturezone(successiveheating scans,notshownhere,arealsodevoidofsuchsharpexothermic transitions), demonstrating the irreversibility of these two transitions.Theonlyeventrecordedduringthecoolingrunisa well-defined

D

CpheatcapacitystepatT=428C,reproduciblein

successiveheating/coolingcycles.

3.2. FT–IRspectroscopyofS4peptidesandS4fibresinthefreeze-dried state

TheFT–IRspectraofS4peptidesandS4fibres(Fig.2)aretypical ofpolypeptidesandproteins,exhibitingtheamideA(freeand H-bonded O–H stretching and N–H stretching), amide I (C O stretching), amide II (C–N stretching and N–H bending), and amideIII(N–Hinplanedeformation)bands.Somedistinctfeatures canbeobservedontheseglobalspectra,particularlythenarrowing andtheshift(from3289to3281cm1_{)ofthemainbandofamide}

AbandinS4fibresaswellasthenarrowingandtheshift(from 1644to1624cm1_{)oftheamideIband.}

TheamideAbandincludesthefreeandbondedabsorptionsof OHandNHgroups[25].Asalreadyobservedin

a

-elastin,theshift towardlowerwavenumbersoftheamideAbandinS4fibresbands islinkedtotheincreaseinthestrengthofthehydrogenbondsand phasestructure[25].

SincetheamideI–IIregionconsistsofseveralbandsstrongly dependentonsecondaryconformations,itwassubjectedtocurve fittinginordertoresolvethevariousunderlyingandoverlapping spectralfeaturesthatcontributetothiscomplexregion[26–30]. The decomposed FT–IR spectra of the amide I–II region are presentedinFig.3.

TheamideIregion(correspondingtothestretchingvibrations oftheC Ogroupofthepeptidebackbone)ofS4peptidescontains a main component at 1633cm1_{, ascribed tobonded}

_b

_turns/

pleated

b

-sheets. Thiscomponent togetherwiththe additional onesobserved at 1694 and 1680 areindicative of anti-parallel

b

-sheetconformationsand

b

turns,respectively[7,27].Sincethe bandscomprisedbetween1640and1660aregenerallyassociated withunorderedconformationsandwaterabsorption[7,24,29],the large componentdetected at 1651cm1_{is certainly due to an}

overlappingofthesecontributions.Itisalsonoteworthythat non-hydrogen bonded C O groups absorb in the 1667–1661cm1

range [7], confirming the presence of PPII conformation in S4 peptide as already revealed in solution by CD [7,29]. The decompositionoftheamideIIbandyieldstwomaincomponents

Fig.1.DSCthermogramsofS4peptidesinsolution(2mM)(heatingandcooling

at1552and1518cm1_{,whichareconsideredrepresentativeofthe}

presenceofthe

b

-sheetconformation[7].Thebandat1537cm1

couldbetentativelyassignedtotheunorderedcomponent[31]. WeproposeforS4peptidesinthefreeze-driedstateadominance of

b

-structurestogetherwithunorderedandPPIIconformations. TheamidIregionofS4fibresisverydistinctfromtheamidI regionofS4peptides,withaprominentcomponentat1627cm1

ascribed tointermolecular

b

-sheets in cross

b

-structures [32], togetherwithabandofincreasingintensityat1697cm1_,whichis

typicaloftheanti-parallel

b

-sheetconformation.Thetwominor bands at 1651 and 1669cm1_{can be attributed to unordered}

conformations/waterabsorption[7,29]and

b

turns[26], respec-tively.Thepredominanceofthecross

b

-structureinS4fibresis attestedbythepresenceoftwomaincomponentsintheamideII region at 1552 and 1525cm1_{, that are representative of the}

b

-sheetconformation[7,32].

3.3. ThermogravimetricanalysisofS4peptidesandS4fibresinthe freeze-driedstate

Thermogravimetric (TG) and temperature/derivative (DTG) plots of freeze-driedS4 peptidesand freeze-dried S4fibresare showninFig.4.Themassdecrementduringtheheatingprocess was determined from TG curves and the temperature of the maximumspeedoftheprocess(Tmax)wasdeterminedfromthe

maximumoftheDTGcurves.TheglobaltrendofthetwoTGplots corresponds to the classical thermal behaviour of freeze-dried proteins [33,34]. The first stage (between 25 and 2008C) is generallyconnectedwiththeevaporationofwaterabsorbedtothe

protein,andcorrespondsto8.5and1.36%ofthetotalmassforS4 peptidesandS4fibres,respectively,meaningthatfreeze-dryingof S4fibresismoreefficientthanfreeze-dryingofS4peptides.

The second stage occurring between 200 and 5008C is associated with the degradation of the sample, namely the progressivedeamination, decarboxylationand depolymerization arisingfromthebreakingofpolypeptidebonds.Thedegradationof S4peptidesconsistsoftwowell-markedstepsasshownbytwo maxima ontheDTG plots at Tmax1=235 and Tmax2=2998C, in

contrastwiththedegradationofS4fibresthatoccursinanarrow temperaturerangewithasinglemaximumatTmax=305.38C.

3.4. WatersorptionexperimentsonS4peptidesandS4fibresinthe freeze-driedstate

Watersorptionexperimentsat53%RHwereperformedat258C (Fig.5)inordertoobtaininformationonthewater/polypeptides affinityfirstrevealedbyTGA.

Undertheseconditionsofhumidity,wecannoticethatS4fibres canadsorbupto85%ofwaterincontrasttoS4peptidesthatcannot adsorb more than 19% of water. As revealed by TGA, it is noteworthythatthefreeze-dryingprocedureismoreefficienton S4fibresthanonS4peptides,leadingtoinitialfreeze-driedstates with2%and8%ofwater,respectively.

Fig.3.DecomposedFT–IRspectrumofS4peptides(A)andS4fibers(B)intheamide I–IIzone(boldline:experimentalcurve;finelines:decompositionintoGaussian peaksandsumoftheGaussianpeaks).

Fig.4.TGAthermograms(TG)andtemperaturederivative(DTG)thermogramsof freeze-driedS4peptidesandfreeze-driedS4fibers.

3.5. DSCanalysisofS4peptidesandS4fibresinthefreeze-driedstate Thermogramsoffreeze-driedS4peptidesandfreeze-driedS4 fibreswererecordedintheheatingmodebetween5and1208C (firstrun)andbetween5and2258Conthesuccessiveruns(Fig.6). Inthecaseoffreeze-driedS4peptides,thebroadendothermic peakobservedonthefirstscanat63.18Cmaybeconsideredasthe first order transition commonly observed in a broad class of hydratedbiopolymers.Byanalogywithpreviousworks[33,35,36], thisendothermiceventisattributedtotheevaporationofbound water, andthe valueoftheenthalpy(

D

H=135J/g)iscoherent withanamountofboundwatercomprisedbetween8and10%as measuredbothbyTGAanddehydrationoverP2O5at1058C.Onthe

second thermogram, the specific thermal answer of wholly dehydratedS4peptidescanbeobserved.Thepresenceofaglass transitionat147.38C(

D

Cp=0.45J/g/K),reversibleonsuccessive

scans,underlinesthefactthatfreeze-driedS4peptidespossessan amorphous region, witha lack oflong-range order, likeelastin

[37,38]. As already observed by TGA, the degradation of S4 peptidesbeginsabove2108C.

In the case offreeze-dried S4fibres, theendothermic event associatedwiththeevaporationofwaterisweaker(

D

H=27.9J/g) than the event reported for S4 peptides. The ratio between enthalpies is roughly the same as the ratio between the total amountsofboundwatermeasuredbyTGAanddehydrationover P2O5at1058C.

The main event detected on the second thermogram is the sharp endothermic peak at 145.68C associated to an order!disorder transition, which can be considered as to the thermal signatureof amyloidsfibres.Thesharpnessof thisfirst ordertransitionisindicativeofahighlycooperativeprocess.This transition,notobservedonthethirdscan,isirreversible. 3.6. DSCanalysisofS4fibresinthehydratedstate

Freeze-driedS4fibres5mginweightwereequilibratedover MgN2O66H2Osaturatedsolutionsat258C(relativehumidity53%)

untilthewateruptakereachedamaximum.Thetotalhydration levelofthesampleswasevaluatedbygravimetry,andwasequalto 84.3% under these conditions of humidity. Different hydration levelswereobtainedequilibratingthefullyhydratedsamplesover KOH saturated solutions at 258C(relative humidity8%)during differenttimes, allowingagradual dehydration ofthesamples. Completedehydrationwasobtainedbyaheatingat608Cduring

10min. The thermograms of the differently hydrated samples recordedintheheatingmodeareshowninFig.7.Forthesakeof clarity,onlyonecoolingthermogram(correspondingtothesample containing84.3%ofwater)hasbeensuperimposedinthisfigure. Forhighhydrationlevels,apartofwaterabsorbedinthesample isabletocrystallizeasshownbytheexothermiceventsat–148C and–398ConthefirstcoolingthermogramoftheFig.7.Onthe successiveheatingthermogram, themelting ofthiscrystallized watergivesrisetoendothermicphenomenabetween–30and08. Thismeltingpeakconsistsoftwocontributionsforhighhydration levels,suggestingthepresenceoftwokindsoficeinthiscase.For lower hydration levels (70%, 58% and 29.8%), only the low temperature peak is observed; in previous studies on water insolublebiologicalpolymers,thispeakwasattributedtofreezing boundwater[39],correspondingtolooselyboundwateradsorbed onpolymerhavinga certainkindofcrystallinestructure which maybethesameasthestructureofnaturalice[40].Thesecond important event detectable on the thermograms of differently hydratedS4fibresistheendothermicpeakoccurringbetween15 and358C,forhydrationlevelscomprisedbetween84.3and2.1%, vanishingforcompletelydehydratedsamples,andindicativeofthe transitionfromanorderedtoanunorderedstructure.

4. Discussion

4.1. ThermalbehaviourofS4peptidesinsolution–irreversible aggregation

In the case of S4 peptides, the first heating thermogram reported in Fig.1 gets an insightinto thepossibleaggregation mechanismintoS4amyloidsfibresevidencedinapreviouswork byturbidimetryandbiochemicaltests[7].Firstly,thepositive

D

Cp

contributionat378Ciscertainlyassociatedwiththeprogressive destabilizationofthepolyprolineII(PII)conformationfrom0to 608Casitwasevidencedbycirculardichroism[7],shiftingthe equilibrium between PPII, unordered structures and

b

turns towards

b

turnspreferentialconformation.Itmustbepointedout thatPPIIisaflexiblestructuredevoidofintramolecularhydrogen bondsthatcaneasilyinterchangewithotherconformations[7].

Comparingthespecialfeatureofthesharpexothermicpeaks occurringat61and788CwiththeDSC-monitoredaggregationof insulin[41],

b

-lactoglobulin[42]and

b

2-microglobulin[43]into amyloidfibres,weproposetoascribethemtotheformationof

b

-sheets and cross

b

-structures, leading to the irreversible

Fig.6.DSCthermogramsoffreeze-driedS4peptidesandfreeze-driedS4fibers (first,secondandthirdheatingruns).

Fig.7.DSCthermogramsofS4fibersatvarioushydrationlevels(coolingand heatingruns).

aggregation of the S4 peptidesinto S4 fibres. This multi-step, irreversibleaggregationmainlydrivenbyhydrophobic interac-tionsinvolvesbothformationofhydrogenbondsinto

b

-sheets structures [41,42], dipoles–dipoles interactions into the steric zipper and breakdown of hydrogen bonds (rearrangement of wateraroundthepeptide,expulsionofwateratthe

b

-sheet–

b

-sheet interface of the steric zipper); the total area of the exothermic peaks is estimated at –6J/g (where the mass corresponds tothemassof dryS4peptides); thisnetnegative enthalpyiscertainlyduetoatotalincreaseoftheglobalhydrogen bondsnetworkdensityandthepositivecontributionofVander Waalsinteractionsatthedryinterfaceofthestericzipper[16–18]. Thisassumptioniswellsupportedbyturbidimetryexperiments, suggestingforS4peptides anirreversibleinverse-temperature phase transition above 608C. The reorganization of water molecules around the

b

-sheets structure could explain the associatedvariationsoftheheatcapacity,sincetheheatcapacity changeoriginatesprimarilyfromthehydrogen-bonding proper-tiesofwaterneartheproteinsurface[44,45].

4.2. ConformationofS4peptidesandS4fibres–relationwiththermal stability

OurFT–IRresultsingoodagreementwiththestructuraldataon amyloidfibrils;whileS4polypeptidesadoptawidedistributionof conformations (PPII, unordered conformations,

b

-sheets and

b

turns),S4fibresconsistmainlyofintermolecular

b

-sheetsincross

b

-structures,withanincreasingstrengthoftheinvolvedhydrogen bonds.

ThemultiplicityofthethermaldegradationevidencedbyTGA on S4 peptides can be correlated with the multiplicity of the secondary structures revealedby FT–IRanalysis,the unordered structurebeinglessstablethanthehighlyordered,highhydrogen bonded

b

-sheets. Moreover, it is noteworthy that even the degradationofthemorestablecomponentinS4peptidesoccurs atlowertemperature(T=2998C)thaninS4fibres.Thisthermal stabilizationof

b

-sheetsintheS4fibrescanberelatedtotheir organization into cross

b

-spines with steric zippers, with a densificationofhydrogenbonds.

4.3. PhysicalstructureofS4peptidesandS4fibresinthefreeze-dried state

TheamorphouscharacterofS4peptidesisevidencedbyDSC withthepresenceofaglasstransitionwithoutanyendothermic peakthatwouldbeindicativeofanorderedstructure.Thislackof long-range order is due to the coexistence of unordered conformations,labile

b

turnsandPPIIconformationsasevidenced byFT–IRanalysisinthedrystate.Itisnoteworthythattheglass transitiontemperatureofS4peptidesisclosetotheglasstransition temperature of thepolypeptideencoded by exon10 of human tropoelastin (Tg=144.28C, Mw=2100.5g/mol) [10], and lower

than recombinant freeze-driedhumantropoelastin (Tg=1908C)

[26]orfreeze-driedelastin(Tg=2008C)[30].

Inagreementwithvibrationalandthermogravimetricresults, thethermaltransitionsofS4fibresareverydistinctfromtheS4 peptidesones.Thesharpendothermicpeakat145.68C,indicative ofthecooperativemeltingofalong-rangeorderedstructurecanbe addressed to the thermal signature of amyloid fibres, forming highlyorderedcross

b

-structureswithintra-andintermolecular

b

-sheets thatextend throughout theentire lengthof thefibre. SuchanendothermiceventhasalreadybeenobservedbyDSCin the hydrated state for the fibrils of the N47A mutant of the R-spectrinSH3[13].Thetemperatureofthispeakisindicativeof animportantstabilizationoftheordered

b

-sheetsstructurewhen S4formsfibres.Itmustbepointedoutthattheenthalpyofthis

endothermicevent(

D

H=4J/g)isinthesamerangeoforderthan freeze-driedcollagentypeI[33,46],whichcanbealsoconsidered asa‘‘crystalline’’biopolymerpossessingaverylong-rangeorder. 4.4. PhysicalstructureofS4fibresinthepresenceofwater

Thefirstimportantpointconcernsthedifferenceofsolubilityof S4peptidesandS4fibers:ifS4peptidesaresolubleinwaterlikea wide class of linear polypeptides, once formed S4 fibres are insolubleandcannotbestudiedinsolution.Thatisthereasonwhy S4 fibres were analyzed in this work at different degrees of hydration.

TheverydistinctwateraffinityofS4peptidesandS4fibresin thefreeze-driedstaterevealedbywatersorptionexperimentsis quite striking at first glance because of the same chemical composition ofboth samples.Froma polymer pointof view, it can appear paradoxical since S4 peptides possess unordered conformations in contrast to S4 fibres that are quasi-wholly organized in ordered cross

b

-structures. Interestingly, a large amountofwatercanbetrappedinthishighlyorderedstructure,as alreadyobservedforothersystems,suchashydrogels[40], multi-lamellarvesicles[47]andsilk-keratincomposites[48]constituted of numerous interstices,bringing to theforedifferentstates of water(freezingfreewater,freezingboundwaterandnon-freezing boundwater).

InthecaseofS4fibres,thetrappingofalargequantityofwater mustbeconnectedtothedefinitionofthewetinterfacesbetween

b

-sheetsof the outside facesof thepair of sheets revealedby structuralanalysisofamyloidfibres[16].Asamatteroffact,there are two distinctly different interfaces between

b

-sheets of the cross

b

-spine:adry,dewettedinterfacebetweenthepairedsheets engagedintothestericzipper,incontrasttothehighlyhydrated externalfacesexposedtosolvent[16].

ThecontentofcrystallizedwaterintodifferentlyhydratedS4 fibreswasestimatedfromtheheatingthermograms(Fig.8)asthe ratioofthemeltingenthalpy

D

Hofthehydratedsampleandthe meltingenthalpyofbulkwater[49](333.55J/g)andreportedin

Fig. 8.By difference, theuncrystallizedwater amountwasalso evaluated and reported in Fig.8. In contrast tothe amountof crystallized water that increases with the hydration level, the contentofuncrystallizedwaterreachesamaximumforhydration levelscomprisedbetween30and70%.Inthisrangeofhydration, approximatively15%ofwatermoleculesaretightlyboundtoS4 fibres.We havealsoreportedinFig.8theenthalpy(

D

H fibres)

Fig.8.AmountsofcrystallizedanduncrystallizedwaterinS4hydratedfibersand enthalpyofthesecondendothermicpeakasafunctionofthetotalhydrationofS4 fiberscomputedfromFig.7.

normalizedtothemassofdryfibres,correspondingtothesecond endothermic event detected between 15 and 358C on the thermograms of Fig.7, associated to anordered structure. The enthalpy of this event sharply increases between 0 and 5% of hydration,meaningthatuncrystallized/boundwater isessential forthestabilizationofthisstructure.Interestingly,theenthalpyis maximumwhentheamountofuncrystallizedwaterismaximum, confirmingthisassumption.ItisclearlyshowninFig.8thatan excess of crystallized water destabilizes this ordered structure, resulting alsoina decrease ofbound water.Water, inthis case possessesapeculiarbehaviour,verydistinctfromwhatobservedfor globularproteins[49],wheretheamountofuncrystallizedwater increaseswithincreasingwatercontent,reachingamaximumfor highhydrationcontents.Thisendothermiceventcanbeconsidered asthethermalanswerofthewetinterfaceofthecross

b

-fibres, mainlyduetothedisruptionofhydrogenbondsbetweenwaterand polypeptidicchainswhenthetemperatureincreases.

5. Conclusion

Incontrasttotropoelastinthatcoacervatestogiverisetonative fibrillarstructures,withanequilibriumattheconformationallevel between unordered conformations, labile PPII and

b

turns,

b

-strands and

a

-helices, essential for its biological function, the synthetic derived-elastin peptide S4 mimicking the released fragment from elastolysis by MMP-12 self-assembles into

b

-structure-rich amyloid-like fibres [7], which shows a greater degree of conformational restriction than the native elastin structure. Inthiswork,weattemptedtogetaninsightintothe physicalstructureandwateraffinityofthispeptideinitslinear stateand itsaggregatestate,sincefew studiesintheliterature have reported thermodynamics of amyloid fibreformation and thermalcharacterizationofamyloidfibres.

Firstly, it was shown that the formation of irreversible aggregates when temperature is raised up in S4 polypeptide solutioncouldbemonitoredbyDSC,corroboratingtheprevious turbidimetryassays.Thismulti-stepmechanismisassociatedwith anetnegativevariationofenthalpy,duetoanetpositiveincrease ofH-bondsandVanderWaalsinteractions.Furtherexperiments performedatdifferentconcentrationsanddifferentscanrateswill benecessarytoquantitativelyestimatethebalancesofchangesin enthalpyandentropy.Moreover,itwillbeessentialtoevaluatethe influenceofthemedium(inpeculiartheroleofcalcium)onthis aggregationphenomenoninordertocorrelateitwithpathologic conditions.

Inthecondensedstate,thethermalsignatureofS4precipitated fibresiscompletelydistinctfromtheoneofthelinearpolypeptides; theformerishighlystableandordered,whilethelatterislessstable, withoutanylong-rangeorder.Theseradicallydistinctfeaturesatthe ultrastructural level arewell correlated withthe majorchanges detectedattheconformationallevelbyFT–IRanalysis.

Finally, DSC is well suited toevidence the peculiar thermal signatureofthewetinterfacesoftheoutsidefacesofthepairof sheetsstructureofamyloidsfibrilsfirstproposedbyNelsonetal.

[16]onstructuralanalysisofelongatedmicroscrystals. References

[1]ChungJH,SeoJY,LeeMK,EunHC,LeeJH,KangS,etal.Ultravioletmodulationof humanmacrophage metalloelastaseinhumanskininvivo.JInvDermatol 2002;119:507–12.

[2]VaradiDP.Studiesonthechemistryandfinestructureofelesticfibersfrom normaladultskin.JInvDermatol1972;59:238–46.

[3]FanK,NagleWA.Amyloidassociatedwithelastin-staininglaminaraggregates inthelungsofpatientsdiagnosedwithacuterespiratorydistresssyndrome. BMCPulmMed2002;2:5.

[4]Doostkam S, BohlJR, SahraianA,MahjoorAA. Amyloiddepositsinsenile vertebralarteries,immunohistologicalandultrastructuralfindings.Pakistan JBiolSci2008;11:1852–5.

[5]TaddeseS,WeissAS,NeubertRH,SchmelzerCE.Invitrodegradationofhuman tropoelastinbyMMP-12andthegenerationofmatrikinesfromdomain24. MatrixBiol2010;22(Suppl1):E56–66.

[6]TamburroAM,PepeA,bochicchioB,QuaglinoD,RonchettiIP.Supramolecular amyloid-likeassemblyofthepolypeptidesequencescoded byexon30 of humantropoelastin.JBiolChem2005;280:2682–90.

[7]bochicchioB,PepeA,DelaunayF,LorussoM,BaudS,DauchezM. Amyloidogen-esis of proteolytic fragments of human elastin. J Royal Soc Chem 2013. doi:10.1039/b00000000x.

[8]LuanCH,ParkerTM,ChanneGowdaD,urryDW.Hydrophobicityofaminoacid residues: differential scanningcalorimetry and synthesisof the aromatic analoguesofthepolypentapeptideofelastin.Biopolymers1992;32:1251–61. [9]Rodriguez-CabelloJC,AlonsoM,PerezT,HerguedasNM.Differentialscanning calorimetrystudyofthehydrophobichydrationoftheelastin-based polypen-tapeptide. Poly(VPGVG), from deficiency to excess ofwater. Biopolymers 2000;54:282–8.

[10]TintarD,SamouillanV,DandurandJ,LacabanneC,PepeA,BochicchioB,etal. Humantropoelastinsequences:dynamicsofpolypeptidecodedbyexon6in solution.Biopolymers2009;91:943–52.

[11]MegretC,GuantieriV,LamureA,pieraggiMT,LacabanneC,tamburroAM. Phasetransitionsandchaindynamics,inthesolidstate,ofapentapeptide sequenceofelastin.IntJBiolMacromol1992;14:45–9.

[12]Rodriguez-CabelloJC,AlonsoM,DiezMI,CaballeroMI,HerguedasNM. Struc-turalinvestigationofthepoly(pentapeptide)ofelastin,poly(GVGVP),inthe solidstate.MacromolChemPhys1999;200:1831–8.

[13]MorelB,VarelaL,Conejero-LaraF.Thethermodynamicstabilityofamyloid fibrilsstudiedbydifferentialscanningcalorimetry.JPhysChemB2010;114: 4010–9.

[14]SamouillanV,DandurandJ,NasareL,BadimonL,LacabanneC,Llorente-Corte´s V.Lipidloadingofhumanvascularsmoothmusclecellsinduceschangesin tropoelastin protein levels and physical structure. Biophys J 2012;103: 532–40.

[15]HeiseH,CelejMS,BeckerS,RiedelD,PelahA,KumarA,etal.revealsstructural differencesbetweenfibrilsofwild-typeanddisease-relatedA53Tmutanta -synuclein.JMolBiol2008;380:444–50.

[16]NelsonR,SawayaMR,BalbirnieM,MadsenAØ,RiekelC,GrotheR,etal. Structureofcross-bspineofamyloid-likefibrils.Nature2005;435:773–8. [17]NelsonR,EisenbergD.Recentatomicmodelsofamyloidfibrilstructure.Cur

OpinStructBiol2006;16:260–5.

[18]EspositoL,PedoneC,VitaglianoL.Moleculardynamicsanalysesofcross-b -spinestericzippermodels:b-sheettwistingandaggregation.PNAS2006; 103:11533–8.

[19]XuW,SuH,ZhangJZ,MuY.Moleculardynamicssimulationstudyonthe molecularstructuresoftheamylinfibrilmodels.JPhysChemB2012;116: 13991–9.

[20]CheonM,ChangI,HallCK.SpontaneousformationoftwistedAb16-22fibrils inlargescalemoleculardynamicssimulations.BiophysJ2011;101:2493–501. [21]Serpell LC. Alzheimer’s amyloid fibrils:structure andassembly. Biochim

BiophysActa2000;1502:16–30.

[22]ToyamaBH,WeissmanJS.Amyloidstructure: conformationdiversityand consequences.AnnRevBiochem2011;80:552–85.

[23]SchleegerM, vandenAkkerC, Deckert-Gaudig T,DeckertV, Velikov KP, KoenderinkG,etal.Amyloid:frommolecularstructuretomechanical prop-erties.Polymer2013;54:2473–88.

[24]Hu X, KaplanD, Cebe P. Determiningbeta-sheet crystallinity in fibrous proteinsbythermal analysisandinfraredspectroscopy. Macromol2006; 39:6161–70.

[25]PopescuMC,VasileC,Craciunescu O.Structuralanalysisofsomesoluble elastinsbymeansofFT–IRand2DIRcorrelationspectroscopy.Biopolymers 2010;93:1072–84.

[26]HuX,WangX,RnjakJ,WeissA,KaplanDL.Biomaterialsderivedfrom silk-tropoelastinproteinsystems.Biomaterials2010;31:8121–31.

[27]DebelleL, AlixAJP,WeiSM,JacobMP, HuvenneJP,BerjotM,etal.The secondary structure and architecture of human elastin. Eur J Biochem 1998;258:533–9.

[28]BonnierF,RubinS,DebelleL,VenteoL,PluotM,BaehrelB,etal.FT–IRprotein secondarystructureanalysisofhumanascendingaortictissues.JBiophoton 2008;1:204–14.

[29]DyksterhuisLB,CarterEA,MithieuxSM,WeissA.Tropoelastinasa thermody-namicallyunfoldedpremoltenglobuleprotein:theeffectoftrimethylamine N-oxideonstructureandcoacervation.ArchBiochemBiophys2009;487:79–84. [30]SalviAM,MoscarelliP,SatrianoG,BochicchioB,CastleJE.Influenceofamino acidspecificitiesonthemolecularandsupramolecularorganizationof gly-cine-richelastin-likepolypeptidesinwater.Biopolymers2011;95:702–21. [31]TamburroAM,PanarielloS,SantopietroV,BracalelloA,BochicchioB,PepeA.

Molecularand supramolecularstructural studiesonsignificantrepetitive sequencesofresilin.ChemBioChem2010;11:83–93.

[32]LopesDHJ,MeistertA,GohlkeA,HauserA,BlumeA,WinterR.Mechanismof isletamyloidpolypeptidefibrillatinatlipidinterfacesstudiedbyinfrared reflectionabsorptionspectroscopy.BiophysJ2007;93:3132–41.

[33]SamouillanV,Dandurand-LodsJ,LamureA,MaurelE,LacabanneC,GerosaG, etal.Thermalanalysischaracterizationofaortictissuesforcardiacvalves bioprostheses.JBiomedMatRes1999;46:531–8.

[34]SionkowskaA,Skopinska-WisniewskaJ,GawronM,KozlowskaJ,PlaneckaA. Chemicalandthermalcross-linkingofcollagenandelastinhydrolysates.IntJ BiolMacromol2010;47:570–7.

[35]Puett D.DTA andheatsofhydrationofsome polypeptides.Biopolymers 1967;5:327–30.

[36]MegretC,LamureA,pieraggiMT,LacabanneC,GuantieriV,tamburroAM. Solid-statestudiesonsyntheticfragmentsandanaloguesofelastin.IntJBiol Macromol1993;15:305–12.

[37]HoeveCAJ,FloryCAJPJ.Theelasticpropertiesofelastin.Biopolymers1974;13: 677–86.

[38]SamouillanV,DandurandJ,LacabanneC,HornebeckW.Molecularmobilityof elastin:effectofmoleculararchitecture.Biomacromolecules2002;3:531–7. [39]McCrystalCB,FordJL,Rajabi-SiahboomiAR.WaterdistributionStudieswithin

celluloseethersusingdifferentialscanningcalorimetry1.Effectofpolymer molecularweightanddrugaddition.JPharmSci1999;88:792–6.

[40]HatakeyamaH,HatakeyamaT.Interactionbetweenwaterandhydrophilic polymers.ThermochimActa1998;308:3–22.

[41]DzwolakW,RavindraR,WinterR.Hydrationandstructure:thetwosidesof theinsulinaggregationprocess.PhysChemChemPhys2004;6:1938–43. [42]StirpeA,RizzutiB,PantusaM,BartucciR,SportelliL,GuzziR. Thermally

induceddenaturationandaggregationofBLG-A:effectoftheCu2+

andZn2+

metalions.EurBiophysJ2008;37:1351–60.

[43]Sasahara K,YagiH,NaikiH,GotoY.Heat-induced conversionofbeta 2-microglobulinandhenegg-whitelysozymeintoamyloidfibrils.JMolBiol 2007;372:981–91.

[44]CooperA.Heatcapacityofhydrogen-bondednetworks:analternativeviewof proteinfoldingthermodynamics.BiophysChem2000;85:25–39.

[45]KinoshitaM,YoshidomeTJ.Molecularoriginofthenegativeheatcapacityof hydrophilichydration.JChemPhys2009;130.144705-1/11.

[46]MilesCA,GhelashviliM.Polymer-in-a-boxmechanismforthethermal stabi-lizationofcollagenmoleculesinfibres.BiophysJ1999;76:3243–52. [47]KaasgaardT,MouritsenOG,JørgensenK.Freeze/thaweffectsonlipid-bilayer

vesiclesinvestigatedbydifferentialscanningcalorimetry.BiochimBiophys Acta2003;1615:77–83.

[48]LeeKY,HaWS.DSCStudiesonboundwaterinsilkfibroin/S-carboxymethyl keratineblendfilms.Polymer1999;40:4131–4.

[49]PanagopoulouA,KyritsisA,SabaterI,SerraR,GomezRibellesJL,ShinyashikiN, etal.GlasstransitionanddynamicsinBSA-watermixturesoverwiderangesof compositionstudiedbythermalanddielectrictechniques.BiochimBiophys Acta2011;1814:1984–96.