Beating the Heat - Fast Scanning Melts Silk Beta Sheet Crystals.

0.71 MB·English

by  Cebe, Peggy

Beating the Heat - Fast Scanning Melts
Silk Beta Sheet Crystals
SUBJECTAREAS:
PeggyCebe1,XiaoHu2*,DavidL.Kaplan2,EvgenyZhuravlev3,AndreasWurm3,DanielaArbeiter3{
SOFTMATERIALS
&ChristophSchick3
BIOMATERIALS
BIOMATERIALS-PROTEINS
1DepartmentofPhysicsandAstronomy,MedfordMA,02155,USA,2DepartmentofBiomedicalEngineeringTuftsUniversity,
BIOPHYSICS MedfordMA,02155,USA,3InstituteofPhysics,UniversityofRostock,18051Rostock,Germany.
Received Beta-pleated-sheetcrystalsareamongthemoststableofproteinsecondarystructures,andareresponsible
16August2012 fortheremarkablephysicalpropertiesofmanyfibrousproteins,suchassilk,orproteinsformingplaquesas
inAlzheimer’sdisease.Previousthinking,andtheacceptedparadigm,wasthatbeta-pleated-sheetcrystalsin
Accepted
thedrysolidstateweresostabletheywouldnotmeltuponinputofheatenergyalone.Hereweoverturnthat
19December2012 assumptionanddemonstratethatbeta-pleated-sheetcrystalsmeltdirectlyfromthesolidstatetobecome
randomcoils,helices,andturns.Weusefastscanningchipcalorimetryat2,000 K/sandreportthefirst
Published
reversiblethermalmeltingofproteinbeta-pleated-sheetcrystals,exemplifiedbysilkfibroin.Thesimilarity
24January2013
betweenthermalmeltingbehavioroflamellarcrystalsofsyntheticpolymersandbeta-pleated-sheetcrystals
isconfirmed.Significanceforcontrollingbeta-pleated-sheetcontentduringthermalprocessingof
biomaterials,aswellastowardsdiseasetherapies,isenvisionedbasedonthesenewfindings.
Correspondenceand
requestsformaterials
shouldbeaddressedto
Manystructuralproteins,inthesolidstate,containextremelystablebetapleatedsheets(Fig.1).These
P.C.(peggy.cebe@
secondary structures are stabilized by inter-chain hydrogen bonding in which every amino acid is
tufts.edu)orC.S. laterally bonded twice to its nearest neighbor chain segments. The hydrogen bonds are responsible
(christoph.schick@ fortheextraordinaryphysicalpropertiesofseveralnaturalproteins,likesilknotedforitshightensilestrength
uni-rostock.de) (0.5,1.3 GPa),toughness(63104,163104 J/kg)1–4,biocompatibility,andbiodegradability.Theyalsoenable
theformationofinsolublebeta-pleated-sheetstructuresinmanydisease-formingproteinssuchthosethatform
plaques,asinAlzheimer’sdisease.Thepresentpioneeringinsights,whichdemonstratethefeasibilityofthermal
*Currentaddress: meltingofbeta-pleated-sheets,suggestalternativeprocessingstrategiesfortheclassoffibrousproteins,andeven
DepartmentofPhysics possiblyfornoveldiseasetherapies,relatedtobeta-pleated-sheetformingproteinswhichcouldbemeltedbylaser
&Astronomy,and fastheating.Thisworksuggestsnewwaystothermallyprocesssolidproteinsintonovelmaterialforms,usefulfor
Departmentof biomaterials.Theabilityreversiblytochangethebeta-pleated-sheetcrystalcontentinfibrousproteinsbyfast
thermaltreatmentswillhaveanimpactonmanyaspectsofbiomaterialpropertiesthatneedtobecontrolledfor
Biomedical
optimalstructure-functionrelationships,including,e.g.,mechanicalproperties,thinfilmprocessing,andcon-
Engineeringand
trolleddrugreleasebehavior.Biologicaloutcomes,relatedtoratesofdegradabilityinvivo,andresponsesofcells
Sciences,Rowan
tosilkmaterialscaffoldsbothcriticallydependuponbeta-pleated-sheetcrystalcontent2,5,6.Forexample,lower
University,Glassboro,
beta-pleated-sheetcrystalcontentresultsinmorerapidratesofremodelingofsilk-basedbiomaterialsinvivoas
NJ08028,USA. wellastheresponseofstemcellsgrownonthesefibrousproteinsubstrates6,7.Growthofstemcellscouldalsobe
directedalongapatternedsubstrate,createdbyselectivemeltingofbeta-pleated-sheetcrystalsonthesubstrate.
{Currentaddress: Theabilitytocontrolthiscrystalphasetransitionbyareversiblethermalprocesshaspotentialapplicationsinthe
InstituteforBiomedical fieldsofsoftmaterials(e.g.,ultrathinfilms,hydrogels,microspheres,nanoparticles,nanofibers,orcomposites),
controlled drug release and delivery, biosensors, biophotonics, nano-biotechnology, and tissue regeneration.
Engineering,
Beta-pleated-sheet crystalcontentisdirectly correlated, forexample, toreleasekineticsofmodelcompounds
UniversityofRostock,
ordrugssequesteredinsilkbiomaterials8.
18051Rostock,
Previousthinking,andtheacceptedparadigm,wasthatunlikehelicalsecondarystructuresorbetastrands
Germany.
whichcandenatureinsolution,thesebeta-pleated-sheetcrystalsinthedrysolidstatecouldnotmeltuponinput
ofheatenergyalone.Thefastheatingmethodsemployedinthepresentstudyprovethatevenbeta-pleated-sheet
crystaldominatedstructuralproteinslikesilk,willthermallymeltundertheappropriateconditionsassketchedin
Fig.1candverifiedbyFTIRspectroscopy.
Hereweusesilkasanexemplarystructuralprotein.Similartosyntheticpolymerlamellarcrystals,thebio-
molecularsilkchainundergoeschainfoldingtoformtwo-dimensionalsheetswhichstacktogethertoformthree-
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Figure1|SilkFibroinStructureBeforeandAfterAssumedThermalMelting. (a)Athree-chainsilksequenceisdepictedbasedontheaminoacid
hexamer,GAGAGS,withanti-parallelmolecularchainaxes.Thechaincomprisesatomsofcarbon(gray),nitrogen(blue),oxygen(red),andhydrogen
(white),whichhaveintermolecularhydrogenbonding(representedasthreeshortverticallines).(b)Themolecularchainsstacktogethertoformthe
three-dimensional,betapleatedsheetcrystal.Thissecondarystructureisformedbyspidersandsilkwormsnaturallyduringfiberspinningfromthe
solutionstate,whichprocessconvertsrandomcoils,turns,andhelicesintothree-dimensionalcrystals.(c)Thetemperaturevs.timeprofileisshown
duringfastthermaltreatment,whichcausesthebetapleatedsheetcrystals(atpointA)tobecomemeltedwithinafewtenthsofasecond,reversingthe
naturalfiberspinningprocess,andrestoringthematerialtothenon-crystallinesolidstate(atpointB).Theon-chipmeltingusesheatingandcoolingrates
ofthousandsofdegreespersecond.
dimensionalcrystals(Fig.1b).IntheB.moridomesticatedsilkworm, and stability that makes silk useful in garments and in surgical
beta-pleated-sheetcrystalsareinducedtoformnaturallyduringthe sutureshasalsoimpededeffortsbyscientiststostudyitsformation
process of silk fiber spinning from water solution in the salivary andexplain itsproperties. Attempts to meltthebetapleated sheet
gland5,9–15. Silk fibroin protein reconstituted from degummed silk crystalsthatcomprisesilk16havealwaysresultedindecomposition
also crystallizes into beta-pleated-sheet crystals from concentrated whichobscuresthemeltingprocess(Fig.2a).Inorderfullytochar-
water solutions, or from the dry state upon exposure of the non- acterizefibrousproteinslikesilk,knowledgeoftheirthermalbeha-
crystallinefibrointomethanol(MeOH) ortohotwater vapor5,9,10. viorisessential.Recently,heatcapacitymeasurementswereusedto
Onceformed,thebeta-pleated-sheetcrystalsstabilizethestructure investigatetheglasstransition(T )andcrystallization(T)processes
g c
andgivesilkitsuniquephysicalproperties.Theremarkablestrength insilkfibroin16–18,andshowedthesimilarityofthesetwotransitions
Figure2|ComparisonofThermalCharacteristicsofSilkandSyntheticPolymeratDifferentHeatingRates.Observabletransitionsduringfirst(1,red)
andsecond(2,blue)heatingincludeglasstransition(T),crystallizationexotherm(T),meltingendotherm(T ),andthermaldegradation(T ).
g c m d
Endothermsarepresentedwithdownwarddeflection.(a)FilmofreconstitutedB.morisilkfibroinproteinduringDSCheatingat2K/min.Thesample
degradesandlosesmassuponfirstheating.Thesecondheatingscandoesnotretracethefirst,andnoglasstransitionorcrystallizationcanbeobserved.
(b)Syntheticpolymer(exemplifiedbyisotacticpolystyrene,iPS)duringDSCheatingat2K/min.Sampledoesnotdegradeafterheatingto538K,andthe
overlappingsecondheatingtraceshowsthesamethermaltransitions.(c)FilmofreconstitutedB.morisilkfibroinprotein,nowheatedat2,000K/susing
fastscanningchipcalorimetry,showsmeltingofbetapleatedsheetcrystals.Thesecondtrace,aftercoolingfromthemeltat2,000K/s,confirmsthenon-
crystallinenatureofthefilmafterbeta-pleated-sheetsweremeltedduringthefirstscan.(d,e)Samplefrom(c),imagedatroomtemperaturewith
unpolarizedlightusinga203objectiveontheflat,opticallytransparentsensor,before(d)andafter(e)fastheatingto650K.Asaresultofmeltingthe
filmchangesshapeslightly.
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tothoseseen inflexible-chain syntheticpolymers. However,when overlapped curve 2, indicating that no thermal decomposition has
performingheatflowmeasurementsattherelativelyslowratesof1– occurred.
20 K/min,typicalofdifferentialscanningcalorimetry(DSC),thesilk ThedataofFig.2cwereobtainedusingverysmallsamplemasses,
melting transition (T ) is obscured by decomposition16–18, even on the order of tens of nanograms heated on a chip calorimeter
m
thoughthemeltingtransitionisseeninpolymersheatedundersim- (Supplementary Fig. S3). The chip calorimeter contains heating
ilar conditions. Thermogravimetric studies of silk heated at 2 K/ and temperature sensing elements and gives a direct measure of
min12undernitrogengasatmosphereshowedthatthermaldecom- the sample’s heat capacity through analysis of the differential
positionbeginsat470 K,andcontinuestooccuroverabroadtem- power22–25 (Supplementary Figs. S3–S7). All fast scanning experi-
perature range7, causing mass loss and converting the silk to black ments are conducted under an inert nitrogen atmosphere. In the
char(SupplementaryFig.S1,insetimages).Correspondingheatflow centerofthechipsensor,thesilkfilmissupportedon,500 nmthin
dataofdrysilk(SupplementaryFig.S2,step4drying)undernitrogen SiNxmembranecoveredbyasimilarlythickSiO2layer,whichallows
gas atmosphere (Fig. 2a, and Supplementary Fig. S1) shows that infraredradiationtobetransmittedthroughthesampleforinsitu
endothermic heat flow also occurs along with decomposition (sig- spectroscopic analysis after different stages of thermal treatment.
nified by T ). The sample loses 25% of its mass by the time the The chain conformation in the crystals yields particular bands in
d
temperaturehasreached548K(SupplementaryFig.S1).Incontrast, the Fourier transform infrared (FTIR) spectra allowing detection
an unambiguous crystal melting transition occurs in polymers with ofcrystallinityevenintheseverysmallsamples16,17,26,27.
flexiblemolecularchains,suchaspolystyrene(Fig.2b),polyolefins,or TheSiNxmembraneisalsoopticallytransparentanddirectoptical
nylons.Butinsilk,beta-pleated-sheetcrystalmeltingcannotbesepa- imagingofsampleswasperformedbeforeandafterfastheating,as
rated from degradation using DSC, even though silk has flexible showninFig.2d,e,respectively.Themicroscopywasperformedat
molecularchainsandisachemicalanaloguetonylon-2. roomtemperature,in-situ,whilethesampleremainedinplaceonthe
Theinterchain bondingin fibrousproteins issostrongthat the sensor.Silkfibroin filmscontainedalargefraction, 0.30–0.4016,of
intrachaincovalentbondsbegintodegradewellbeforethemelting beta-pleated-sheetcrystalsbutneverthelesswereopticallytranspar-
transition. As a result, important information about the thermal ent. This suggests the beta-pleated-sheet crystals are in the nan-
properties of silk, such as the temperature range of the beta sheet ometer size scale, and therefore do not scatter visible light. Silk
crystal melting, is unable to be assessed using conventional DSC fibroin films were found to adhere slightly to the sensor. Visual
heatingrates.Itwasourthinkingthatwecouldmitigatethedegra- indicationofmeltingcanbeseeninsmoothingofthecutfilmedges
dationprocess,whichoccursevenunderaninertgasenvironment, andsomefilmshrinkage(Fig.2d,e)orformingadropletoutofsilk
byemployingaveryfastheatingrate19,suchasratesnowavailable fiberswithlossofbirefringence(Figs.3d,eand4a,b).Moreimpor-
throughuseofchipcalorimetry(Fig.1c).Then,itwouldbepossible tantly,thesilkfilmsandfibersafterfastheatingto650 Khavenot
beenblackenedbydecomposition.
to observe the thermal melting of beta-pleated-sheet crystals and
providefundamentalinformationthatwouldcompletetheanalogy Weusedaseriesofthermaltreatments(Fig.3)todemonstratethat
betweensyntheticpolymerthermaltransitionsandthoseoffibrous meltingandcrystallizationforsilkfibroinwerefullyreversible.The
samplewasheatedandcooledonthechipsensorat2,000 K/swhile
proteins.
heatflowdatawerecollected.Beforeandafterfastheating,thesec-
ondarystructureofthesamplewasexaminedin-situusingFourier
Results
transform infrared spectroscopy (FTIR) (Fig. 3a)16 of the spectral
Thetechniqueoffastscanningchipcalorimetryallowsasampletobe regionsofAmideI(1600,1700 cm21,assignedtothepeptideback-
heatedandcooledextremelyrapidly,withheatingrates19–21andalso bone)andAmideII(1500–1600 cm21)26,27.Secondarystructurewas
coolingrates22–25ofmanythousandsofkelvinpersecond,providinga correlatedwiththetreatmentconditions(SupplementaryFig.S8).
temperaturevs.timeprofileillustratedinFig.1c(seeSupplementary
First, initially non-crystalline film (Fig. 3a, curve M0) contains
Fig.S2).Inchipcalorimetry,timeofexposureofasampletohigh onlyhelices,turns,randomcoilsandsidegroupsecondarystructures
temperaturesisgreatlyreducedsothatanentireheat/coolcycleis inAmideI,butnobeta-pleated-sheetcrystals.IntheAmideIIregion,
completedin,0.3 s.Throughthistechnology,weobtainheatflow the Tyrosine ring vibration at 1515 cm21 increases after drying,
dataofproteinswithinfractionsofasecondduringheatingtoavery whichwereportedpreviouslyforthicksilkfibroinfilms16.Thesame
hightemperature(.700 K),preventingthermaldecompositionthat samplewasthenexposedaltogetherthreetimestoMeOH(bysoak-
occursformostproteinsattemperaturesbeginningaround,500 K. ingtheentiresensor1sampleinavesselcontainingMeOHandthen
This provides us a unique opportunity to investigate the melting dryinginair)toinducetheformationofthebeta-pleated-sheetcrys-
behavior of solid protein crystals in the high temperature region tals,andeachexposurewasthenimmediatelyfollowedfirstbyinsitu
(500–700 K). FTIRspectroscopytoverifythepresenceofbeta-pleated-sheetcrys-
Thermalanalysisoffibroinusingfastscanningchipcalorimetryis talsandbyopticalmicroscopytodocumentshapeandcolorofthe
showninFig.2c(redcurve1).Silkfibroincontainingbeta-pleated- sample, and then by heating at 2,000 K/s in the rapid scan calori-
sheet crystals, when heated in an inert atmosphere at 2,000 K/s, metertomeltthejust-formedbeta-pleated-sheetcrystals.Afterthe
displaysitsglasstransitionatTg(,474 K,atthisheatingrate)fol- rapid calorimetric heating scans the state of the sample was again
lowedbyacompletemeltingendothermatTm(endothermpeakisat checked by FTIR spectroscopy and optical microscopy. Without
,530 K).Coolingthefilmatthesamefastratepreventstheoccur- removingthesamplefromthesensorthiscyclewasrepeatedthree
renceofanyre-crystallizationofthebeta-pleated-sheetsandacom- times.
pletelynon-crystallinesampleresults(Fig.1c).Whenthissampleis Absorption spectra after crystallization (i.e., after exposure to
heatedasecondtime(Fig.2c,bluecurve2)theheatflowoverlapsthe MeOH) but before melting (Fig. 3a, red curves C1–C3) show the
first scan at both low and high temperature. The heat flow trace characteristicvibrationalbandoftheantiparallelbeta-pleated-sheet
showsareductionofT (to,467 K)andaslightlylargerstepincre- crystalsat1629 cm2116.Thecrystallinesamplewasthenheatedto
g
mentatT (becausethereisnowmorenon-crystallinematerialpre- 650 Ktomeltthecrystals,cooled,andexaminedagainwithFTIR.
g
sent)andnomeltingendotherm(becausetherearenomorebeta- Absorptionspectra(Fig.3a,bluecurvesM1–M3)revealabsenceof
pleated-sheetcrystalspresent).Becauseofthehighheatingratealso the1629 cm21characteristicbeta-pleated-sheetvibration,confirm-
nocrystallizationisseen(thetimethesampleisinthetemperature ingthatthesilkfibroinbetapleatedsheetshavebeenmeltedbythe
range where crystallization can occur is too short). This film was rapid heating to 650 K, and furthermore, they do not recrystallize
heatedandcooledrepeatedlyat2,000 K/s,andallsubsequentscans uponcoolingbecauseoftheextremelyfastcoolingrate.Thefilmsare,
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Figure3|ComparisonofStructuralandFastScanningCalorimetricData. RoomtemperatureFTIRabsorbancespectraofsilkfibroin(a)with
correspondingcalorimetricdataat2000K/s(b).(a)Initialnon-crystallinesilk(M0).Silkexposedin-situthreetimestoMeOH(C1–C3red:18h–C1;
2.5h–C2;20min–C3)crystallizes,andbeforefastheatingcontainsbeta-pleated-sheetcrystals,withcharacteristicbetasheetabsorbancemarkedat
1629cm21.Afterfastheatingto650K,beta-pleated-sheetcrystalsmeltandonlyrandomcoils,turns,andalphahelicesremain(M1–M3blue).(b)Heat
capacityvs.temperatureat2000K/sforthesamefilmshownin(a),presentedasmatchedsets.Initialnon-crystallinesilk(M0).MeOH-exposed
crystallinesamples(C1–C3red)duringfirstheating.Aftercooling,thefilmwasimmediatelyreheated(M1–M3blue).Absenceofmeltingendothermsin
reheatingscansconfirmssilkisnolongercrystalline.(c)Heatcapacityvs.temperatureat2000K/s,forwaterannealedsilkfibroinfilmandabundleof
nativedegummedcocoonfibers.DownarrowmarksT;uparrowmarksonsetofmelting;horizontalblackarrowmarksregionoffibermeltingandshape
g
changetodropletmorphology.(d,e)Abundleofdegummedcocoonfibers,imagedinunpolarizedlightat203magnificationbefore(d)andafter(e)
heatingto690K.Theseparatefiberscontractwhenthebeta-pleated-sheetcrystalsmelt,removingthephysicalcrosslinks,andallowingthefibersto
coalescenceintoasingledroplet.
however,fullycapableofcrystallizingonceagain,eitherbyexposure 10–20K/min used in commercial differential scanning calorimeters.
toMeOHortohotwatervapor(Fig.3a).Theseexposuretreatments Similarlyforsilkfibroinprotein,thesecond,andanysubsequent,mea-
result in lowering of the glass transition temperature ("plasticiza- suringcyclesat2,000K/sshowedthecharacteristicsofanon-crystalline
tion"), thus providing sufficient mobility to the fibroin molecular sample:theonlyfeatureobservedintheheatcapacitytraceisthewell-
chainssothatcrystallizationcanoccur17,18.Afterexposure,thesam- pronounced glass transition step change. No thermal decomposition
plesweredriedfreeofwater(orMeOH)byheatingthemabovethe peakwasobservedintheheatcapacity,andasaresultoftheabsence
glasstransitiontemperature(SupplementaryFig.S2,step4). ofdecomposition,thesubsequentscansalloverlappedeachother.The
Companionthermalscansofthisfibroinfilmtakenat2,000 K/s thermaldatademonstrateforthefirsttimethatthebeta-pleated-sheet
areshowninFig.3bforthesamesetoftreatments.Heatcapacity crystalsinsilkfibroinfilmscanbereversiblycrystallizedandthermally
traces show the behavior of the initially non-crystalline film (blue melted,andthemeltingtemperaturerangeisfrom510–560K.
curve M0) and then the now-crystalline film after its exposure to Thefastheatingtechniquewasalsoappliedtoasmallbundleof
MeOH(redcurvesC1–C3).Eachscanofthecrystallinefilmshowsa degummed native cocoon silk fibers, heating them at 2000 K/s
meltingendothermoccurringabovetheglasstransitiontemperature. (Fig.3c).(Optical images ofafiberbundle areshown inFig.3d,e
Superimposed on the first heating scan is a second heating (blue andforasinglefiberinFig.4).Theonsetofmeltingofthenative
curvesM1–M3)showingheatcapacitycharacteristicofnon-crystal- fibersoccursatamuchhighertemperature(,610 K)thantheonset
linematerial.Thesecondscandisplaysnomeltingendotherm,and ofmeltingofasolventcrystallizedsilkfibroinproteinfilm(,530 K),
confirmstheFTIRresultthatnobeta-pleated-sheetcrystalsremain regardlessofwhetherthefilmwaswater-annealed(Fig.3c)orMeOH
inthefibroinfilmaftermeltingtheminthefirstheatingscan. treated(Fig.3b).Atthesametimethatbeta-pleated-sheetcrystals
Uponrapidcoolingat2,000 K/stoatemperaturebelowtheglass aremelting,thehighlyorientedfibersinthebundleundergomolecu-
transition,thejustmeltedfilmremainsnon-crystalline.Thecooling larretraction.Thereasonfortheretractionisthatthebeta-pleated-
andheatingratesweresufficientlyrapidtopreventrecrystallization sheetcrystalsactasthermo-reversiblecrosslinksinthefiber.Once
of molten silk protein during the heat-cool cycle. This behavior is thecrystalsmelt,thefiberisabletoretractfromitsas-spunaligned
analogoustothatofsyntheticpolymershavingglasstransitiontem- condition,intorandomcoilconformationandasinglemoltendrop-
peratures well above room temperature: (e.g., poly(etheretherke- letforms(Fig.3e,Fig.4b).
tone), PEEK, T 5 418 K (145uC)28; isotactic polystyrene, iPS, Before melting, FTIR shows that the degummed cocoon fiber
g
T 5 373 K (100uC)29: cooling from the molten state prevents compriseshighlyorientedbeta-pleated-sheetcrystals(degreeofcrys-
g
the polymer from crystallizing even at the relatively slower rates of tallinity,0.62–0.6530).Asistypicaloforientedcrystallinefibers,the
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chain,previouslyonlyaqueousororganicsolvent-basedprocessing
ofsuchbeta-pleated-sheetcrystaldominatedproteinscouldbeused
tounderstandandcontrolcrystallizationbehavior.Withthefindings
reportedhere,nowthermalprocessingcanalsobeconsideredasa
processingapproach.Thisfindingremovesakeydistinctionbetween
biologically-derivedvs.syntheticpolymersandallowsnewthinking
about materials engineering even from beta-pleated-sheet crystal
enrichedstructuralproteins.
Silksaretheprototypicalbeta-pleated-sheet-containing proteins
innature.Mostotherproteinsutilizebeta-pleated-sheetstocontrol
protein structure and function, and as such, understanding self-
assemblyandunfoldingofthevariousstructuralstatesandtransi-
tionsusingrapidandcontrolledthermalheating,wouldaffordnew
insightsintothebiophysicsandbiochemistryoftheseproteinsys-
tems. Since reversible thermally-induced melting transitions have
beendemonstratedforsilk,thethermo-analyticalmethodsusedin
thisworkcannowbeappliedtoanybeta-sheet-containingprotein,
wheresuchsecondarystructuresregulatethestabilityandfunctionof
proteins.Theabilitytocontrolthebeta-sheetstructuraltransitionsof
proteinsingeneral,viafastscanningcalorimetry,couldpossiblyhelp
clarifythermalmodesofproteinfoldingandunfoldingandoptimize
the understanding of these transitions. Furthermore, an improved
Figure4|SilkStructureBeforeandAfterMelting. Asingledegummed
understanding of thermal melting transitions could have broader
nativecocoonfiberbefore(a)andafter(b)meltingat2000K/s,imaged
implicationsforrulesofproteinself-assembly,towardsproteinpuri-
betweenthepolarizer(vertical)andtheanalyzer(80degreestothe
ficationandrefoldinginthefieldofbiotechnology,forexample,orin
polarizer)at103magnification.Suggestedsecondarystructureis
newwaystothinkaboutregulatingbiocatalysis.
indicatedinthelowerpanels:beta-pleated-sheetcrystalsindegummed
nativecocoonfiber(c)andrandomcoils,turnsandalphahelicesafter
melting(d).Thefiguredemonstratesthemainresultsofthiswork,thatsilk Methods
canbemelteddirectlyfromthesolidstateupontheinputofheatenergy Materials.CocoonsofB.morisilkwormsilk(obtainedfromTsukuba,Japan),were
boiledfor25mininanaqueoussolutionof0.02MNaCO andrinsedthoroughly
alone,withoutdegradation. 2 3
withwatertoextracttheglue-likesericin2,7,9,11,16,17,30,32.Theresultantfluffyfibrous
materialservedasthesamplefordegummednativecocoonsilkfibers.Tocreatesilk
polarizingmicroscopeimageFig.4ashowsabrightandcolorfulfiber proteinfilms,thesilkfibroinproteinfibersweredissolvedina9.3MLiBrsolutionat
60uCfor4,6h,andthendialyzedindistilledwaterusingaSlide-a-Lyzerdialysis
duetothestronglyuniaxialbirefringence.Thatis,theindexofrefrac-
cassette(Pierce,MWCO3500)for2days.Aftercentrifugationandfiltrationto
tionofthemolecularchainsispreferentiallyalignedalongthefiber removeinsolubleresidues,thefinal2,5wt.%silkfibroinaqueoussolutionwascast
axis.Uponinitialheatingto,690 K,thebeta-pleated-sheetcrystals inpolystyrenePetridishestomakesilkfibroinfilms.Afterdryingtoremovewater,
inthefibermeltandthefiberlosesitsorientation(Fig.4b),andhence theresultingdryfilmthicknesswasaround10mm.
Theas-castfilmswerecompletelynon-crystallinepriortotherapidscanchip
itsbirefringence.
calorimetry.Priortoplacementonthechipsensortherewasnosignatureofbeta
pleatedsheetsineithertheFTIRabsorptionspectrumortheWAXSdiffractogram16.
Thedriedlessordered,non-crystallinefilmsrapidlyandcompletelydissolvedwhen
Discussion
placedinroomtemperaturewater,whereasanysemicrystallinefilmsorfibers,
Thefindingsfromthisnewworkhavetwomajorimplications.First, containinginsolublebetapleatedsheets7,11,16,17,32,33,wouldnot.
sincetheearliestreportsofbeta-pleated-sheetstructures31therehave Underanopticalmicroscope,verysmallpiecesofsilkfibroinproteinfilmwitharea
beennoreportedobservationsofthermallyinducedmeltingofdry, typicallylessthanabout1003100mm2werecutfromthelargerparentfilm.By
electrostaticinteraction,thefilmwaspickedupandplacedontotheactiveareaofthe
solid state beta-pleated-sheet crystals. The fast heating methods
chipsensorusingafinewire.Thesameapproachwasusedtomountindividual
employed in the present study prove that even beta-pleated-sheet degummednativecocoonsilkfibers,orsmallbundlesoffibers,ontothechipsensor.
crystal dominated structural proteins like silk, will thermally melt
undertheappropriateconditions. Thermaltreatmentsofthesamples.Samplesofsilkfibroinfilmsweresubjectedtoa
seriesoftreatments(SupplementaryFig.S2)insideoroutsidetherapidscan
Onegoalofourresearchinexploringproteinmeltingphenom-
calorimeterinvolvingallorsome(dependinguponsample)ofthefollowingsteps:
enon is to provide a ‘‘bridge’’ between modern synthetic polymer 1.erasureofthermalhistory,2.crystallizationbysoakingtheentiresensor1samplein
scienceandbiomaterialscience,whichwillbenefitmultiplescientific avesselcontainingMeOHandthendryinginair(orbyexposuretohotwatervapor),
areasandbroadeninterdisciplinaryinteractions.Thesecondmajor 3–4.dryingandrelaxation,and5.multiplethermalscanstohightemperatureat
extremelyfastrates.Allfastscanningwasperformedunderaninertnitrogengas
implication of this work, relating to this goal, is that proteins are
environment.Thedegummednativecocoonsilkfiber,orsmallbundlesoffibers,were
analogoustosyntheticpolymers,evenfromathermodynamicsper- merelydriedandthermallyscanned(i.e.,steps1and2inSupplementaryFig.S2were
spective. Melting phenomena demonstrate that crystallized solid omittedforthenativecocoonsilkfibers).Duringthefirstmeasurementcycleto
proteins,exemplifiedherewithsilkfibroin,canbeconsideredasfully 650Kforsilkfilms(SupplementaryFig.S2,firstcycleofstep5),allofthecrystalline
samplescontainingbetapleatedsheetcrystalsbecamemolten(datashowninFig.3
compatiblewithallmodernpolymertheoriesandexperimentsabout
andSupplementaryFig.S4).Uponcoolingfromthemelttobelowtheglasstransition
crystallizationandmeltingdevelopedinthelastcentury,withpro- temperature,thesamplessolidifiedintonon-crystallinefilms.Thesecond,andall
teinsexpressingthesamethermaltransitionsasthesyntheticpoly- subsequent,measuringcyclesshowedthesamplesremainednon-crystalline,unless
mers, and unfolding from the solid state crystal to the liquid melt onceagainsubjecttocrystallizingtreatments(SupplementaryFig.S2,step2)which
involveexposuretoeitherhotwatervaporormethanol(MeOH),orisothermal
throughtheinputofheatenergyonly.Forthefirsttime,weareable
holdingabovetheglasstransitiontemperature.
tomakeadirectparallelcorrespondencebetweenthethermalbeha-
vior of protein beta-pleated-sheet crystals, and lamellar crystals of FastScanningCalorimetry(FSC).TheFSCmeasurements19–25,34wereperformed
synthetic polymers, identifying the crystal melting transition in usingathinfilmchipsensorXI-399ofXensorIntegration(Netherlands)
addition to the non-crystalline glass transition in both systems. (SupplementaryFig.S3)employingarecentlydevelopeddifferentialpower
compensatedcalorimeter.Detaileddescriptionofthemeasurementprincipleand
Whileproteinsmaybemorecomplexintermsofchemicaldiversity,
instrumentalsetupisgivenelsewhere22,23.Duetothesmallsamplemassandsmall
with 20 different monomers (amino acids) available in the main addendaheatcapacities,ratesashighas1,000,000K/sonheatingandcoolingcanbe
SCIENTIFICREPORTS |3:1130|DOI:10.1038/srep01130 5
www.nature.com/scientificreports
achievedandheatflowtothesamplecanbemeasuredupto100,000K/s.Twosuch 12.Zheng,Y.etal.Directionalwatercollectiononwettedspidersilk.Nature463,
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Additionalinformation
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Nature424,1057–1061(2003). Competingfinancialinterests:Theauthorsdeclarenocompetingfinancialinterests.
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License:ThisworkislicensedunderaCreativeCommons Howtocitethisarticle:Cebe,P.etal.BeatingtheHeat-FastScanningMeltsSilkBetaSheet
Attribution-NonCommercial-NoDerivs3.0UnportedLicense.Toviewacopyofthis Crystals.Sci.Rep.3,1130;DOI:10.1038/srep01130(2013).
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