1

Themaingoalistobeableto“film”aproteininac6on.Theideaistofollowthedifferentstructuralchangesoccurringduringanenzyma6creac6on,anallostericchange,theaccessaligandorsubtratetoabindingsiteorac6vesite,thereleaseofaproductorligandfromtheac6vesiteorbindingsite,etc…Forenzyma6creac6on,theatomicresolu6onisrequired,asweneedtobeabletoassesspreciselyalltheinteratomicinterac6on(hydrogenbonds,saltbridges,vanderWaalscontacts,etc…).Lowerresolu6onmaybeenoughtomonitorglobalstructuralchanges(allostericchanges).Wealsoneedtoobtaininforma6onattherelevant6mescale.Thisreallydependonthesystemstudied.

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The6mescaleatwhichwecanobservestructuralchangesisextremelylargeandbasicallygoesfrom100fs(atomicmovementinducedbyatomicorelectronicexcita6on)uptothesforunfoldingprocesses,thusarangeofatleast13orderofmagnitude.Mostofmacromoleculefunc6onarepossiblethankstolargeramplitudecollec6vemovements.Thepresenceofcollec6vemovementisillustratedbythechangeinslopeatagiventemperature,whenthemeansquaredatomicdisplacement<∆u2>isploQedasafunc6onoftemperature.Onthediagram,weclearlyobservetwodifferentbehaviors,eitherbeloworabove180-200K.Thismeansthatwemayblockcollec6vemo6onbydecreasingthetemperaturebelow180Kandthustraptheproteininagivenstate.Liquidnitrogentemperature(100K)isappropriateforsuchatrapping.

4

Thetwotechniquesthatallowtogetstructuralinforma6onatmosttheappropriate6mescalesareX-raydiffrac6onandSmall(orWide)AngleX-rayscaQering(SAXSorWAXS).Themaincharacteris6csofthetwotechniquesaremen6onedonthisslideandthenextone.

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Howtotriggerareac6on?Oneofthemainissuewillbetotriggerthereac6onofallmoleculeswithinthesampleattheverysame6meinordertohavethereac6ongoingoninaperfectlysynchronouswayonallmolecules.1)  Thediffusionofsubstrate,co-factor,etc…canbedonewithinthecrystal.Itis

possibleinmanycases,sinceproteincrystalsdocontainasignificantamountofsolvent:thevolumeofaproteincrystaloccupiedbysolventmayvaryfrom30to80%,withsome6melargechannelinwhichligandscaneasilydiffuse.Thedrawbackisthatitisarela6velyslowprocess,thatcanbeusedonlyforquiteslowreac6on.Theuseofverysmallcrystals,thankstoalwaysmorepowerfulX-raysourcemayspeedupthetriggering.

2)  Endogenousphotosen6vity.Lightistheperfecttoolstotriggerareac6onasitcantrulyac6vateallthemoleculesatthesame6me.Insomecases,theproteinisendogenouslyac6vatedbylight(presenceofachromophore,protein-ligandinterac6onthatcanbebrokenbyphotons,…

3)  Oneusesacagedcompound,thatdobindclosetotheac6vesite,andthatcanbeuncagedbylight.Inthatcasealso,wecanthustriggerthereac6onatthesame6meinallmoleculesofthesample.

4)  TheX-raysthemselvescaninduceareac6on(radiolysis,foranexample).Inprinciple,itmayworkasgoodaswithlighttriggering,butoneshouldpayaQen6onthatX-raysusedforthestructuralinves6ga6onsarenotalsosignificantlyac6va6ngthereac6on.

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9

Some reminder about classical crystallography. The main message is that the structure obtained represent an average over the data collection time (typically 1 mn, with today’s synchrotron, but can be up to a few hours for less intense X-ray sources) and over all the molecules present in the crystal (1014-1018). The data collection is clearly not fast enough to image an enzymatic reaction.

10

Sameremarkfor“classical”SAXSexperiments:weobtainstructuralinforma6onthatisalsoaveragedoverthedatacollec6on6me(10–20s)andallthemoleculespresentinthesample(typically1015).Alsonotfastenoughbyfarformostenzyma6creac6ons.

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Onpeutbaisserlatempératurepourpiégerlamacromoléculedansunétatpar6culier.Eneffet,lafonc6ondenombreusesprotéineestdirectementliéàl’existencedemouvementscollec6fsdelargeamplitudequisontobservésaudelàd’unecertainetempérature,typiquementdel’ordrede180K.Onpeutdonc“figer”uneprotéinedansunétatpar6culierandlacongelantàlatempératuredel’azoteliquide(100K).Audessousd’unecertainetempérature,lesenzymesnefonc6onnentplus,carlamoléculesnepeutplusfranchirlesbarrièresd’énergied’ac6va6on.Celarevientàdiminuerfortementlaconstantecataly6quekcat,quisuituneloid’Arrheniusenfonc6ondelatempérature.

14

Plusieursstratégiessontenvisageablespourpiégerunétatintermédiaire:1)  Trigger-Freezze:ondéclenchelaréac6on,puisonbaisselatempératureàun

momentprécis(possiblepourlesréac6onssuffisammentlentes)2)  Freeze-trigger:onbaisselatempérature,puisondéclenchelaréac6on,enfixantla

températuredesortequelapremièreétapedelaréac6onsoitpossible,maispaslasuivante.

3)  SteadyState:idemque2)maisonmain6entuneac6va6oncon6nue(permetd’empêcherquel’étatintermédiairesedépeupleàcausedelaréac6onensensinverse)

4)  Lalce-trapping:Leréseaucristallinpeutempêchercertainmouvement,augmentercertainesénergiesd’ac6va6onetdoncbloquercertainesréac6onsdansunétatintermédiairedonné.Icionjouedoncsurlamodifica6ondel’énergied’ac6va6on,etéventuellementsurlatempérature,sibesoin.

15

Onpêcheuncristalquel’onmetdansunesolu6onquidéclenchelaréac6on.Aprèsuntempsdéterminé,onre-pêchelecristaletonlecongèledansl’azoteliquideOnu6lisececristalcongelépourlesétudesstructuralesetspectroscopiquesIlfautquelaréac6onsoitassezlente(minute).Onasouventdesmélangesd’états.Lacombinaisond’étudesstructuralesetspectroscopiquespermetdemieuxiden6fierlesétatsetlesespèceschimiquesprésentesdansl’échan6llon.

16

LaSORestunemetalloenzyme(Fe)quel’ontrouvechezuncertainnombredebactéries(e.g.Treponemapallidum)etarchae(e.g.Pyrococcusfuriosus)sensiblesàl’oxygène.Ellepar6cipeàunevoiealterna6ved’élimina6ondel’ionsuperoxyde,différentedecelletrouvéechezlesbactériesaérobies,quifaitintervenirlasuperoxydedismutase(SOD),lacatalaseetdesperoxydases.Danslesiteac6fdelaSOR,leFerestcoordonnépar4his6dineséquatorialesetunecystéineaxiale.Ici,nousallonsregarderplusendétailuneétudequicombinecristallographieciné6queetspectroscopieRaman(Katonaetal.,2007)pourcomprendrelemécanismeréac6onneldelaSOR.

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L’idéedebaseestdepiégerlecomplexeFe-peroxo.Pourcefaire,ilsuffitdemeQreenprésencelaformeréduitedelaSOR(Fe2+)etH2O2.Cependant,pouréviterlaforma6onderadicauxhydroxyltrèsréac6fs,LaSORestd’abordoxydéeaveclesodiumhexachloroiridate(IV),puismisenprésenced’H2O2(Pourcetravail,c’estlaréac6oninversequiestdéclenchée,maisl’étatintermédiairerecherchéerestelemême).Pourbloquerlaréac6onaustadeoùlecomplexeFe-peroxoestformé,unmutantspécifiqueGlu114Alaestu6lisépourstabiliserunétatintermédiaire.

18

LecomplexeFe-peroxydeestvudans3unitésasymétriquessur4etsaprésenceestconfirméeparspectroscopieRamanensolu6onetincristallo.LaLys48quiformeunesortedecouvercle(lid)ausiteac6festunrésidutrèsconservéchezlesSORetl’onvoiticisonrôledanslastabilisa6ondesmoléculesd’eauetduperoxo.Lescalculsdechimiequan6queparlathéoriedefonc6onnelledensité(DFT)ontpermisdemontrerquelesmonomèresBetDétaientprotonéssurl’oxygènedistalduperoxo.(A)  Lidlocked-open:Lys48estdésordonnée(B)  Lidlocked-closed:Lysinteragitavecleperoxo.Wat10etWat11par6cipentà

l’interac6on.Lachargeposi6veduNH3+alreleperoxoetfragiliselaliaisonFe-

peroxo(C)  Lidlocked-closed:Lys48sedéplaceunpeu.Wat10etWat11aussi.Wat11est

maintenantdansuneposi6onoùellepeutdonnerunprotonàl’oxygèneproximalduperoxo,pourformerH2O2quiseralibéré.C’estcequidis6nguelesSORdesenzymesàhèmes,oùc’estl’oxygènedistalquiestprotonéundeuxièmefois,avecrelargaged’H2Oetcréa6ond’uncomplexeoxo-feryl,toxique.

19

D’aprèslesdifférentsétatsdéterminés,un“film”dumécanismeréac6onnelpeutêtreproposé.Toutefois,lesconstantesdetempsnesontpasaccessibles.

20

Unvueplusdétailléeetconven6onnelledumécanismeréac6onnelproposépourl’élimina6onO2

-parlaSOR.

21

Thevastmajorityof6meresolvedcrystallographicstudieshavebeenperformedusingtheLauediffrac6ontechnique.Insteadofusingamonochroma6cX-raybeamusedforclassicalstructuralinves6ga6on,apolychroma6cbeamisused.Theadvantageistocollectmuchmoredataforeachorienta6onofthecrystal.Therefore,thedatacollec6onrequireslessimagesinordertoobtainacompletedataset.Thedrawbackisthatsomespotsdooverlapwhichmakesthedataprocessingabitmorecomplex.TheconceptofLaue6meresolvedX-raycrystallographystudiesistoac6vatethemoleculesofthecrystalbyalaserpulse,andthentocollectthestructuraldatawithanX-raypulse.ThedelaybetweenthelaserpulseandtheX-raypulsecanbechosenbytheuser,inordertoinves6gatedifferentstatesalongthereac6onpathway.Theminimum6mescalethatcanbeinves6gateddependonthedura6onofeitherthelaserpulseortheX-raypulse:thelongestofbothdefiningtheminimum6mescale.Agoodsynchroniza6onbetweenlaserandX-raypulseisrequired.Onlyreversiblereac6onsareinves6gatedusingthisexperimentalsetup,sinceonecrystalisusedforcollec6ngseveralimages(infactanen6redataset).Thedatacollec6onismadeatroomtemperature,inordertoallowstructuralchangestohappenandthereturntothe“dark”stateforthecollec6onofthenextimage.

22

Illustra6onbytheEwalddiagramofreflec6onscollectedforone“s6ll”image.Foramonochroma6cexperiment,onlythenodesofthereciprocalspace(greyorbluepoints)thatcrossestheEwaldsphere(letsaytheredone,ifdatacollec6onismadeatλmax)willgeneratespotsonthediffrac6onimage.Ifapolychroma6cradia6onisused,e.g.allwavelengthbetweenλminandλmax,allthenodesofthereciprocalspacethatinbetweentheredandthegreencircle(areashadedingrey)willgeneratespotsonthediffrac6onimage:muchmorethanwithamonochroma6cbeam.

23

SchemeoftheexperimentalsetupusedforLaue6meresolvedexperimentsandpictureofsuchasetuponasynchrotronbeam.YoucanseethattheenvironmentofthecrystalispreQycrowded.

24

25

Thetranstocisisomerisa6onreac6onconcernstheC1-C2-C3-C1’dihedral,whichistransinthedarkstate(anglecloseto180°)andcisintheac6vatedstate(anglecloserto0°).Thetrans-to-cisisomerisa6onisthoughttohappenveryrapidly,inthefstops6mescale,accordingtospectroscopicstudies.However,globalstructuralchangesarerequiredforsignaltransduc6onshouldoccuratamuchlonger6mescale.Manyworkshavebeendoneonthisprotein(wewillgothroughsomeoftheminthenextslides),asPYPisgoodcandidateforacasestudy.Butthemainbiologicalgoalwastounveilthestructuralmechanismthatistriggeredbythephotonabsorp6on,thateventuallytellsthebacteriatochangeofdirec6ontoescapeblueradia6ons.

26

Thefirst6meresolvedstructuralinves6ga6onofPYPwasmadealmost20yearsago.The6mescaleis10ms,sinceathat6me,theX-pulsewasgeneratedbyamechanicalshuQer:onecannotgotomuchlower6mescalewithsuchshuQers.Thesamecrystalisusedforabout300images:itresistpreQywelltoradia6ondamageatroomtemperature,whichisnotalwaysthecaseforproteincrystals.

27

Theelectrondensityomitmap(thechromophorehasnotbeentakenintoaccountfortheFcalcandphasecalcula6ontoavoidanypossiblebias)ofpanelCandD(bleachedstate)showsthattwostatesarepresentinthecrystal.Thismeansthatthereac6onhasnotbeentriggeredinallthemoleculesofthecrystal,butonabout50%ofthem.Sincetheresolu6onishighenough(1.9Å),thetwostatescanbeseenandmodeledanyway(yellowforthedarkstate,whiteforthebleachedstate).PanelAandBshowthedifferenceelectrondensitymapFbleached–Fdark.Posi6veregions(blue)indicatewheretomovethechromophorea~erac6va6onandcorrespondtothebleachedstate(whitemodelbuildinit)andnega6veregions(red)indicatethatfulloccupancyofthedarkstateconforma6onofthechromophoreisnotmaintaineda~erac6va6on.

28

Thebleachedstructure(white)correspondtotheI2orpBstateofnextslide.The“dark”conforma6on(yellow)correspondthethegroundstatepG.

29

In2005,sevenyearslater,ansstudy(insteadof10msforthepreviousone)ofthePYPmechanismismadepossiblebytheuseofanslaserandofsynchrotronX-raypulses.Amuchmoredetailedviewofthecyclecanbeproposed,whith4differentstates:Icp(cis-planarintermediate,α),onepRstatewithtwopossibleconforma6ons(β)andtwopBstates(γandδ)andthe6meconstantsassociatedtoeachstate.

30

Accordingtotheelectrondensitychanges(bluerepresentposi6vedifferences,i.e.posi6onwheremoreelectron(atoms)shouldplaced;redrepresentnega6vedifferences,i.e.posi6onswhereatomsshouldnolongerbe,atleastpar6ally),At1ns,thetrans-to-cistransi6onhasalreadyoccurredonthechromophore,butchangesatlimitedtothecloseneighborhoodofthec1-c2-c3-C1’dihedral.Significantchangesofthechromophoreringposi6onoccursattheµsscale,withthetwopRconforma6onobserved.Thechromophoeconforma6onalchangeiscompletedatthemsscale.Butlargerscalemovementhavebeentriggered.Thedisplacementofanhelix,quitedistantfromtheac6vesiteisobservea~er50ms(δstate,panelJ).

31

Here,thedifferentstateshavebeenmodeledintotheelectrondensity(panelsA,B,C,D)andthesuperposi6onwiththeground(dark)stateisshowninthepanesE,F,G,H.Thetwopossibleconforma6onsofthepRstateareshown:oneinwhichtheringhasnotmovedmuch,andoneinwhichithasmoveclosertoposi6onadoptedinpBstate.

32

Asimplerviewofthedifferentstructuraltransi6onsobserved.Theblackarrowsindicatethemovementthatwilloccurtoreachthenextstate.

33

Thankstothese47snapshots,thepopula6onofthedifferentstatescanbeploQedonadiagram,asafunc6onof6me.ThepresentdiagramshowsthatthemechanismofPYPspanatleastfromthenstos6mescale,thepB2state,withchangeintheter6arystructure(movementofanhelix),beingthoughttosignalthephotonabsorp6ontothebacteria.However,itisclearthatsomechangeshavealreadyoccurredatthens6mescale,atthatshorter6mescalesarerequiredtoknowthefullstory.

34

In2012,a6meresolvedstudyat100ps,thesmaller6mescalepossiblewithpresentsynchrotrons,hasbeencarriedoutonPYP.Themainmessageprovidedbytheelectrondensitymapsshownhereisthatthetrans-to-cistransi6onhasalreadyoccurreda~er100ps,confirmingthatthetransi6onoccursveryrapidlya~erphotonabsorp6on.

35

AbeQeriden6fica6onoftheearlystepscanbemade,andaslightlydifferentschemeofthereac6oncycleisproposed.TwopRstatesareproposed,pR1andpR2,thatcorrespondtoabeQer6meresolveddescrip6onofthepRcwtransi6ondescribedinthepreviousstudy.Here,theotherconforma6onofthepRstateisnotobserved.

36

Another100psstudy,in2013,confirmsthattwopathwaysexistsforpRstates,withtwodifferentconforma6ons,similartotheonesproposedinthe2005study.

37

Anothersetofexamplesof6meresolvedstructuralstudiesfocusonmyoglobinorhemoglobin.Here,thegoalistounderstandwhathappenwhenthebondbetweentheiron(inthecenterofthehemegroup)andtheligand(CO,bindingmore6ghtlythannaturalligandsO2,CO2)isbrokenbyphotolysis.Ifallostericchangesareknowntobetriggeredbyligandbindingorunbinding,thekine6cofsuchevents,andthedetailedstructuralmechanismsunderlyingallostericchangesremainedtobeinves6gated.

38

In2003,ans6meresolvedstudyofSpermwhale(Cachalotinfrench)myoglobinhasbeenperformed.Twosnapshots,one3nsa~erphotolysisoftheFe-CObond,one316nsa~erphotolysisoftheFe-CObondhavebeenanalyzed.

39

Theanalysisofthedifferenceelectrondensitymapsshowsthatsignificantchangesoccurreda~er3ns.TheCOisnolongerboundtotheFe(largenega6vedensity,inred).Tworesidues,Tyr29andGln64havemovedsignificantlyfromtheirini6alposi6oninyellowtotheirnewposi6oninblue.TheCOseemstohavemovedtotheXe4posi6on.A~er316ns,nomajorfurtherchangesintheimmediatesurroundingoftheheme.TheCOhasnowmovedontheothersideofthehem,inposi6onXe1

40

Except,COposi6on,themaindifferencedifferencebetweenstructuresat3nsand316nsisobservedquitefarfortheac6vesite.Collec6vedisplacementsareslowerandoccursattheµs6mescale.

41

Onthebasesofthistwosnapshots,ananima6onoftheCOphotolysiscanbeproposedbytheauthors.

42

Scapharcainaequivalvis,akindofclam(palourdeorclaminfrench,ashellfishfromthebivalvesfamily)possessadimerichemoglobin.Asforhumanhemoglobin(whichistetrameric),thishemoglobinundergoanallosterictransi6on,withtwowelliden6fiedstates:theR(relaxed)stateandtheT(tense)state,whichhavedifferentaffini6esforligands.Here,themaingoalistounveilthemainstructuralintermediatesalongthestructuraltransi6on.

43

In2006,a5ns6meresolvedstructuralstudyunveiloveraquitelarge6mescale(5ns–80µs).Asforthepreviousstudy,theelectrondensitymapshowsthat5nsa~erlaserpulse,40%oftheCOmoleculesarenolongerboundtotheFeandhavemovedtoadistalsitelabeledCO*inthefigure.TheFeatomhasalsobeendisplacedbyabout0.4Åtowardstheproximalhis6dine.

44

CeQefigureillustrelemouvementdel’atomedeFer,aucentredel’hème.Dansunepremièrephase(5ns-1µs)l’atomedefersedéplacede0.4Åperpendiculairementàl’hèmeversl’his6dineproximale(posi6on≠deRetT).Peudedéplacementdansplandel’hème:onrestedansl’étatR.Puisaprès1ms,l’atomedeFerevientdansleplan(mouvementperpendiculairementauplan)maissondéplacementdansleplandel’hèmemontrequ’iladoptel’étatTets’éloignesignifica6vementdel’étatR(≈0.9Å).

45

Lacourbeduhautmontrequ’entre5nset200ns,lesitedistalduCO(CO*)estoccupéalorsquelesiteproximalest(engrandepar6e)inoccupé.Apar6rde1µs,lesiteproximalretrouve(quasitotalement)sonCOetlesitedistalleperd.Lechangementdeconforma6ondelaphénylalanineF4estplustardif:peudechangementavant1µs,puistransi6onversle«Tstate».LedépartduCOdéclenchedoncunecascadedechangementsstructurauxquiperdurentbienaprèsleretourduCOàsaposi6onini6ale.

46

Thisanimatedpictureshowsthedifferentstepsfrom5nsto60µs.At60µs,mostofthedifferenceelectrondensityisseenattheinterfacebetweensubunits,possiblyindica6ngaemergingallostericchange,whilethedifferenceelectrondensityintheFeimmediatesurroundingshasbasicallyvanished,indica6ngthattheCOisnowbacktoitsini6alposi6on.ThemaintakehomemessageisthatthestructuralcycleofCOphotolysisonhemoglobincanbedescribedbyseriesofeventsoccurringatdifferent6mescales.TheFe-COphotolysis,whichoccurveryrapidlya~erthelaserpulse(<5ns),triggerstructuralchanges(movementofsidechains,movementofsecondarystructures,rota6onofmonomer)thatcon6nueatthe10µs–ms6mescale,whiletheCOhascomebacktoitsini6alposi6oninlessthat1µs.At60µs,theallostericchangeisfarfrombeingcompleted(0.6°rota6onreachedinsteadof3°forthewholerela6vechangeoforienta6onofthetwomonomersintheTstate,comparedtotheRstate)Fasttriggeringeventcantriggerlonglas6ngstructuralevents.

47

Theexamplespresentedbeforeshowthebest6meresolu6onthatcanbeobtainedwithsynchrotronsX-raysources.However,anewsourceofX-ray,theX-rayfreeelectronlaser,isemerging.Afewarealreadyopera6ngintheworld(Japan,USA)andonewillbeopera6nginEurope(Hamburg)in2017.Themainadvantagesare(i)theextremepeakbrilliancethatcanbeobtained(1035photons/s/mrad2/mm2/0.1%bw;today’ssynchrotronsareoffering1026,andshouldbeabletoreach1033intwoyears),and(ii)theshortX-raypulse:10fsinsteadof100ps:againof4ordersofmagnitudein6mescale.

48

ThankstoaverywellfocusedX-raybeam(≈1µm–100nm)andthehighbrilliance,structuraldatacanbecollectedonverysmallcrystals(1µmto100nmineachdimension).Serialcrystallographyhasthusbeendeveloped.Usingasinglecrystaltoobtainacompletedatasetisnolongerpossible,sincetheX-raydosereceivedbyacrystalistoohigh.Eachcrystalisusedtocollectonlyonediffrac6onimage.Sincethepulseisveryshort(10-100fs),nodamageinducedbytheintensepulseisobservedonthediffrac6onpaQern.Thedamageswilloccuratalarger6mescale,whenthedataarealreadycollected.Tocollectacompletedataset,wethusneedalotasimilarsmallcrystals,thatareinjectedasacon6nuousflowtroughthelaserandX-raybeams.Thissetupenablestoinves6gateirreversiblereac6ons,sinceeachcrystalisusedonlyonce.

49

WithX-FELandserialcrystallography,thechallengeistocollectdatabeforedestruc6on.Aseminalpaperin2000showedthatamolecule,becauseoftheiner6aassociatedtoatomicmovements,cannotexplodebeforeafewtensoffs.Thus,isthepulseisshortenough,itsintensitycanbeashighasneeded.Theyalsoes6matedthatstudyonasinglemoleculeinsteadofacrystalcanbeenvisagedifthebrilliancereachesatleast1040.Wearenotthereyet,butnotthatfarIfprogressinX-raysourcearecon6nuingatthesamerate,wemayhopetogetsuchsourcesin5to10years..

50

OneofthefirststructuralstudydonewithanX-FELsource,onphotosystemI,in2011.Theyexperimentallyconfirmedthataproteincrystalcouldresista700MGydoseifthepulsewasshortenough(<100fs),whileinprinciple,adoseof30MGyisconsideredasthemaximumpossibleformostproteincrystalsstudiedonasynchrotronsource.Ofcourse,serialcrystallographyischallenging,ifoneconsiderthequan6tyofcrystalsneeded,thenumberofimagestobecollectedanddataprocessing.Butthisstudydemonstratedthatitwasactuallyfeasible.Andmuchprogresseshavebeenperformedsince.

51

Anotherstep(theul6mate?)in6meresolvedstructuralinves6ga6onofPYP,at100fsin2016usingX-FEL.Here,theLauetechniqueusedwithsynchrotronisnopossible,astheX-raysaremonochroma6c.However,sincecrystalsareexposedonlyonce,themonochroma6capproachcanbeusedwithoutanyproblems.Thetechnicaldataoftheexperimentsareshownontheslide.Inthisstudy,theauthorshavefirstmadeaposi6vecontrolwithadelayof200nsbetweenlaserpulseandX-raypulsetoconfirmtheresultsobtainedinpreviousstudiesmadewithsynchrotronsources.Thus,thenewsetupisnotintroducingunexpectedar6facts.ThepresenceoftwoRstates,R1(β1andβ2features)andR2(γ1andγ2features),withtwodifferentconforma6onshasbeenconfirmed.

52

Before500fs,thechromophoreremainsintransconforma6on,butslightmovementsareobserved.Thecisconforma6onisclearlyseena~er500fs.Thelife6meoftheelectronicexcitatedstateisafewhundredfemtosecond.Thereturntoelectronicgroundisassociatedtotran-cistransi6on.Ifthereac6oniselectronicallytriggeredinabout100fs,itsthermallydrivena~erwardsonamuchwider6mescale.Theenergyofonebluephoton(2.76eV)isten6melargerthantheonerequiredtomovethechromophore(0.2eV)

53

X-FELX-raysourcescanreallyenabletoexploretheveryearlyeventsofthechromophoreac6va6on,witha6mescalecloseto100fs.Andthe6meatwhichthetrans-cistransi6oncanbedeterminedprecisely.Thesuccessionofelectronicandstructuraleventscanthusbusobservedoveralarge6merange.Asforhemoglobin,understandingthephotoac6va6oncycleofPYPrequirestoinves6gateawiderangeof6mescale.Here,itspansover13orderofmagnitudes,fromthetrans-to-cistransi6onoftheC1-C2-C3-C1’dihedralofthechromophore,in≈500fs,tochangesinter6arystructure(movementsofhelices)thattakeplaceinthe10ms–1s6mescale,thatcaninturntriggerbacteriasignalingpathways.Thankstothesenewtools,thefullcycleamanyreac6onsmaybestudiedandalltheuseful6mescalescannowbeinves6gated.

54

Asmen6onedintheintroduc6on,6meresolvedstudiescanalsobydonebySAXSorWAXStechniques.Thesetupisquitesimilartotheoneusedforserialcrystallography,exceptthatproteinisinsolu6oninsteadofbeingpackedinsmallcrystals.SAXSorWAXSexperimentscanonlyprovideinforma6ononglobalchangesoftheprotein(changesinradiusofgyra6on,shapeforSAXS,changesindomainandsecondarystructuresforWAXS).Sincesolu6onstudiesonlyprovide1Dinforma6on(ascaQeringcurve),insteadof3Dforcrystallography(a3Delectrondensitymap),modelingisrequiredtointerpretchangesinscaQeringcurves.

55

Here,thedifferencescaQeringcurvesSlight(q,Δt)-Sdark(q)atdifferent6messhowthatthemajorvaria6onsoccurfor1.5<q<2.5Å-1,indica6ngchangeswithindomains,i.e.movementofsecondarystructures,inthe3–300ps6mescale.Usingmoleculardynamics,thesechangescanbeexplainedbymovementthetransmembranehelices,asshownonthefigure.

56

Inthis6meresolvedSAXSstudyofbacteriorhodopsin,moderatechangesintheshape(q<0.6Å-1)areobservedbetween360nsand100ms.Structuralmodelingalsosuggestsshi~sinsomeregionsoftransmembranehelices.

57

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