TowhomcorrespondenceshouldbeaddressedFax2155732235EmailduttonmailmedupenneduJPhysChemB101021jp992000vCCC18001571999AmericanChemicalSocietyPublishedonWeb09301999 oftheassembl ID: 376136
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EngineeringOrientedHemeProteinMaquetteMonolayersthroughSurfaceResidueChargeDistributionPatternsXiaoxiChen,ChristopherC.Moser,DenisL.Pilloud,BrianR.Gibney,andP.LeslieDutton*JohnsonResearchFoundation,DepartmentofBiochemistryandMolecularBiophysics,ersityofPennsylania,Philadelphia,Pennsylania19104ed:June17,1999;InFinalForm:August23,1999Wehavedesignedandsynthesizedfour--helix-bundleproteinsthataccommodatehemegroupstoactasmolecularªmaquettesºofmorecomplexnaturalelectron-transferproteins.Thesebundlescanbeorientedatanairwaterinterfaceandtransferredontosolidsurfacestofacilitatetheexplorationofthefactorsthatgovernbiologicalelectrontransfer.Wefindthattheorientationofthesemaquettesonanairwaterinterfacecanbecontrolledbychoosingthedistributionofchargedaminoacidsalongthesidesofthehelicesexposedtowater.Thefour-heliceswereassembledeitherastwosubunits,whereeachsubunitconsistsoftwo-heliceslinkedbyaterminalcysteinedisulfidebond,orasasingle,four-helixcovalentunitconsistingoftwohelixhelixmoleculeslinkedbyaterminalcysteine.Ineithercase,wheneach-helixcontainsbothpositivelychargedlysinesandnegativelychargedglutamates,additionofthehemebindingbundlestoanairwaterinterfacecausesthemtoopenupandlieonthesurfacewith-helicalaxesorientedparalleltotheinterface.Incontrast,whenthepositiveandnegativechargesaresegregatedondifferenthelices(twonegative,twopositive)ofthesinglecovalentfour--helix-bundleunit,thebundlepreserveditsintegrityontransfertotheairwaterinterface.Moreover,thepresenceofhemedictatestheorientationoftheaxesofthebundlewithrespecttothesurfaceplane.The-helicesadoptaparallelorientationintheabsenceofhemeandaperpendicularorientationinthepresenceofheme.Circulardichroism(CD)andultravioletvisible(UVvis)spectroscopysupportedbylineardichroismdemonstratethatthesemolecularorientationsarepreservedinLangmuirBlodgettmonolayerfilmsonsolidsubstratesurfaces.Anovelapproachtothestudyofcomplexnaturaloxido-reductaseenzymesistodesignandsynthesizeproteinswiththeminimalrequirementsfortheassemblyandincorporationofactiveredoxcofactors.Thesedenovodesignedredoxproteinsactasmolecularªmaquettesºtouncovertheengineeringbehindtheassemblyofnaturaloxidoreductasesthatpromotedirected,long-rangeelectrontransfer.Wechoosesequencesstronglyfavoring-helicalfoldingtopresenthydro-phobicresiduesalongonefaceofeachhelixandchargedgroupsontheoppositeface.Hydrophobicforcesdriveassociationofthehelicesintoafour--helixbundleinsolution,whilehistidinesinthebundlecoreligateaddedhemes.Thesehemeproteinmaquetteshaveproventopossessthecharacteristicelectro-chemicalandopticalpropertiestypicalofnaturalMaquettescanbeengineeredtoorganizeonsurfacesbyexploitingthegreatfreedomofdenovodesignandchemicalsynthesistomodifyexposedresiduesandcontroltheinteractions-helices.Organizingmaquettesonsurfacestentiallynotonlyfacilitateselectrochemicalstudies,explorethefactorsthatgovernbiologicalelectrontunneling,butalsoformsthefoundationfortranslatingthesehemeproteinmaquettesintonovelfunctionalbiomaterials.studyofthesurfaceassemblyofthesehemeproteinmaquettesprovidesinformationonthenatureofthevariousinteractionsbetweenproteinsandmembranesorartificialsurfaces.Inapreviousstudy,weexploredtheLangmuir(LB)filmpropertiesofourprototypefour--helix-bundleheme-bindingproteinmaquettes.Weshowedthatthishomodimericprototypehemeprotein(showninFigure1Adissociatesontheairwaterinterfaceintoitsmonomericsubunits,,andorientsits-helicalaxesparalleltothesurface,presumablywiththehydrophobicinterioroftheoriginalfour--helixbundleexposedtotheair.Wealsoshowedthattheadditionofhydrophobictails,suchaspalmiticacid,totheN-terminiofthe(hemeproteininducesreorientationathighsurfacepressuresuchthatthe-helicesadoptanangleperpendiculartotheairwaterinterface.Encouragedbytheobservationthatthepolaritydistributionontheexteriorofthebundlescaninfluencethestructuresontheairwaterinterface,wehavefocusedontheeffectofalteringexteriorchargedistributions;threerelatedsynthetichemeproteinmaquettesare1.(.The(hemeproteinhasbeendescribedinthepreviousstudy.Itisahighlywater-solublefour-bundlehemeproteinthatisbasedontheprototypedescribedbyRobertsonetal.,conservativelymodifiedtoimprovepackingofthebundleinterior(seeMaterialsandMethods).Inthebundle,apairofidentical-helicesarelinkedbyadisulfidebondattheircysteinylN-terminitomakeansubunit.Apairofsubunitsspontaneouslyassembleinaqueoussolutiontoformanoncovalentfour--helixbundle(Ononesideofeach-helixtherearesevenglutamates,sixlysines,andonearginineformingalternatepositiveandnegativepatches,whichformacandycanestripedpatternontheexterior *Towhomcorrespondenceshouldbeaddressed.Fax:(215)573-2235.E-mail:dutton@mail.med.upenn.edu.J.Phys.Chem.B10.1021/jp992000vCCC:$18.001999AmericanChemicalSocietyPublishedonWeb09/30/1999 oftheassembledfour--helixbundle.Figure1Ashowsthatthefour--helixbundlecanadoptasynorantitopologyinwhichthedisulfidebondsareatthesameoroppositeendsofthebundle,dependingonthesequenceandcofactorbinding.Thestructureofthisfour--helixbundlehasbeenstudiedbyNMRspectra.2.(.Theaminoacidsequenceofeach-helixofthe(hemeproteinisidenticaltothatofthe(hemeprotein.However,asshowninFigure1B,tworepeating-helixaminoacidsequenceswithsixglycinesbetweenaresynthesizedasasinglesequence.ApairofsinglesequencearelinkedbyadisulfidebondattheircysteinylN-terminitoformacovalentfour--helixbundle(.Theexternalchargesof(and(aresimilar.3.(.The(hemeproteinisavariantofthe(hemeproteinwithamajorchangeinthedistributionoftheexteriorchargedresiduescomparedwith(and(.In(,14glutamatesareplacedinonehelixand14lysinesintheotherhelixtoformahighlyasymmetricallycharged,asshowninFigure1C.Then,aswiththe(,apairofarelinkedbyadisulfidebondattheircysteinylN-terminitoformafour--helixbundleThisreportexploresthepropertiesofthethreehemeproteinmaquettesinaLangmuirBlodgettfilmbalanceandafterdepositiononplanarsubstratesurfaces.Weshowthatthe(and(hemeproteinsdisplaysimilarpropertiesontheairwaterinterfacewhilethe(hemeproteinisdramaticallydifferent.Itisclearthatthesegregatedchargedistributionof(increasesthefour-integritythroughstrongelectrostaticinteractionsbetweenthehelicesandprovidesabasicmaquettedesignadaptedforbothsolutionsandinterfaces.MaterialsandMethodsPeptideSequences.1.(.Theaminoacidsequenceofeach-helixinthe(hemeproteinisHistidineatposition10isforhemebinding.Apairofidentical-helicesarelinkedbythedisulfidebondattheircysteinylN-terminitomakeansubunit.Twospontaneouslyassembleinaqueoussolutiontoformafour-helixbundle(.ThispeptidediffersfromtheprototypeH10A24intheinteriorresiduesL6I,L13F,andA24Fand,asshownbyNMR,hasasingularstructure.2.(TheaminoacidsequenceoftheinthehemeproteinisApairofsinglesequencearelinkedbyadisulfidebondattheircysteinylN-terminitoformafour--helixbundle(3.(Theaminoacidsequenceofthethe(hemeproteinisApairofarelinkedbyadisulfidebondattheircysteinylN-terminitoformafour--helixbundle(ChemicalsandSolvents.Hemin,trifluoroaceticacid(TFA),dimethylsulfoxide(DMSO),andoctadecyltrichlorosilanewerepurchasedfromAldrichChemicalCo.(Milwaukee,WI).Ethanedithioland1-hydroxybenzotriazolwerepurchasedfromFluka(Ronkonkoma,NY).FMOC-protectedaminoacidper-fluorophenylesterswerepurchasedfromPerSeptiveBiosystems(Framingham,MA)withtheexceptionofFMOC-OPfp,whichwasobtainedfromBachem(KingofPrussia,PA).NovaSynPR-500resinwaspurchasedfromCalbiochem-Novabiochem(LaJolla,CA).Allotherchemicalsandsolventswerereagentgrade.WaterwaspurifiedusingaMilli-QwatersystemfromMilliporeCorp.(Bedford,MA).PeptideSynthesis.Thepeptidesweresynthesizedonacontinuous-flowMilligen9050solid-phasesynthesizerusingstandardFMOCBuprotectionstrategywithNovaSynPR-500resinat0.2mmolscale.Thesidechainprotectinggroupsusedareasfollows:Lys(tBoc),Glu(OtBu),Cys(Trt),andArg(Pmc).Thepeptideswerecleavedfromtheresinandsimultaneouslydeprotectedusing90:8:2trifluoroaceticacid/ethanedithiol/waterfor2h.Crudepeptideswereprecipitatedandtrituratedwithcoldether,dissolvedinwater(0.1%TFA),lyophilized,andpurifiedtohomogeneitybyreversed-phaseC18HPLCusingaqueousacetonitrilegradientscontaining0.1%(v/v)TFA.Theresultingpeptideidentitieswereconfirmedwithlaserdesorptionmassspectrometry. Figure1.Designandconstructionofhemeproteinmaquettes.PartAshowstheconstructionofheme;thesolid-phasepeptidesynthesisproducedthe31-amino-acidpeptide,whichisoxidizedtoformadihelixunit.Twounitsself-assembleintoafour-helixbundle(.Thefour-helixbundlecoordinateshemesbybis-histidylligationtoformthehemeproteinmaquetteheme.PartBshowstheconstructionofheme;thesolid-phasepeptidesynthesisproducedthe63-amino-acidpeptide,whichisoxidizedtoformthefour-helixbundle(.Thefour-helixbundlecoordinateshemesbybis-histidylligationtoformthehemeproteinmaquetteheme.PartCshowstheconstructionofheme;thesolid-phasepeptidesynthesisproducedthe63-amino-acidpeptide(withlysinesandglutamatessegregatedondifferenthelices),whichisoxidizedtoformthefour-helixbundle(.Thefour-helixbundlecoordinateshemesbybis-histidylligationtoformthehemeproteinmaquette,hemeJ.Phys.Chem.B,Vol.103,No.42,1999Chenetal. HemeProteinMaquettePreparation.1.HemeThe31-amino-acidpeptidesweredissolvedin100mMam-moniumhydrogencarbonatebuffer(pH9.2)inairtoallowthecysteinestooxidizetoformthe62-amino-acidpeptider4hthesolutionwasfrozenandlyophilized.Whenneeded,peptidesweredissolvedinphosphatebuffer(50mMphosphate,100mMNaCl,pH8.0)toyieldafinalconcentrationof0.02mM(.Hemewasincorporatedinto(toformhemefromastocksolutionof4mMofFe(III)protoporphyrinIX(heme)inDMSObysuccessiveadditionsof0.1hemeperbindingsiteuntil1hemeperbis-histidinesitewasreached.Duringeachaddition,thesolutionwaswellstirredandthenallowedtoequilibratefor5min.ThefinalconcentrationofDMSOintheaqueoussolutionwasalwayslowerthan1:100(v/v).ThehemeincorporationwasmonitoredbytheincreaseoftheSoretbandmaximumbetween411and412nm(120000M2.HemeThe63-amino-acid-speptidesweredissolvedin100mMammoniumhydrogencarbonatebuffer(pH9.2)inairtoallowthecysteinestooxidizetoformthe126-amino-acidpeptide(.After4hthesolutionwasfrozenandlyophilized.Whenneeded,the(peptidesweredissolvedinphosphatebuffer(50mMphosphate,100mMNaCl,pH8.0)toyieldafinalconcentrationof0.02mM(.Hemewasincorporatedinto(toformwiththesameproceduredescribedabove.3.HemeThe63-amino-acid-spep-tidesweredissolvedinhighionicstrengthphosphatebuffer(50mMphosphate,1.5MNaCl)atpH4.4toyieldafinalconcentrationof0.04mM-s,andthenthepHwasadjustedto8.0byaddingNaOH.Thepeptidesolutionwasthenexposedtoairtoallowthecysteinestooxidizetoformdisulfidebondsand(.Hemewasincorporatedinto(toformhemefollowingthesamepro-ceduredescribedabove.SolutionMolecularWeightDetermination.SizeexclusionchromatographywasperformedonaBeckmanSystemGoldHPLCsystemwithadiodearraydetectorusingaSupelcoSigmachromGFC-100column(300mm57.5mm)elutedwithaqueousbuffer.Thecolumnwasstandardizedusingaprotinin(6.5kDa),ribonucleaseA(13.7kDa),chymotrypsin-ogen(25.0kDa),ovalbumin(43.0kDa),andbovineserumalbumin(67.0kDa).LangmuirFilmPreparationandIsothermMeasurement.ALangmuirBlodgettfilmbalance(LaudaFilmbalanceFW2;Sybron/Brinkmann,Westbury,NY)wasusedtomakethefilmsaccordingtoageneralproceduredescribedelsewhere.aqueoussubphasecontained1mMphosphateatpH8.0.Allfilmsweremadeunderanargonatmosphereatasubphasetemperatureof22C.Proteinswerespreadattheairinterfaceusingtheªglass-rodºmethod.Thisinvolvedplacinga0.6mmdiameterglassrodatasmallangle()relativetothetroughplane,justtouchingthewatersurface,towhich2mLaliquotsofmaterialwereaddedevery1015s.Duringthisprocedurethetroughsurfaceareawasheldconstant.Surfaceareaisothermsweremeasuredusingabarrierspeedof10cmSubstratePreparation.Quartzslides,1mminthickness(EscoProducts,OakRidge,NJ),weresonicatedfirstindetergentsolutionandtheninwater.Thiswasfollowedbyimmersioninafreshlypreparedsolutionof4:1(v/v)9598%sulfuricacid/30%hydrogenperoxide(CAUTION:Thissolutionishighlyoxidizinganderyacidicandshouldbehandledwithextreme)atroomtemperatureforabout1h.Theslideswerethenthoroughlyrinsedwithwaterandfinallystrippedofvisiblesurfacewaterwithastreamofargon.Thecleanedslideswerethenrenderedhydrophobicbysilanationwithoctadecyltri-chlorosilane.Thiswasachievedbysonicatingtheslidesfor20minina0.1%octadecyltrichlorosilaneinasolutionof80%hexadecane(99%pure),12%carbontetrachloride,and8%chloroform.Theslideswerewashedthreetimesinchloroformforatleast5minforeachwash.Thequalityofthehydrophobiccoatingwascheckedbyplacinga20mLdropletofwaterontheslide.ThecoatingwasconsideredsatisfactoryifthedropletstartedmovingfreelyandcontinuouslyontheslidewhenthetiltangleoftheglassexceededBlodgettMonolayerFilmPreparation.maquetteswerespreadasdiscussedabove,compressed,andheldataconstantsurfacepressureof20mN/m.Ahydrophobicallymodifiedquartzslidewaspassedverticallyintothesubphaseat3mm/min.Thesubmergedslidewasthenreleasedintoavialrestingatthebottomofthetrough.AfterLBdeposition,theslidewastransferredfromthevialtoaquartzcuvetteforspectroscopiccharacterization.Theentireprocesswasdoneinbuffersothattheslidewasneverexposedtoair.CircularDichroismSpectropolarimetry.CirculardichroismspectraweremeasuredinanAviv62DScirculardichroismspectropolarimeter.Rectangularquartzcuvettesof0.2cmpathlengthwereusedformeasuringsolutionspectra.Forfilmspectra,fourquartzslidescoatedwithLBfilmwerepositionedinaquartzcuvetteof1.0cmpathlengthusingtwoTeflonblocks(eachhasfourparallelslitsandfitstightlyonthebottomandtopofthecuvette)holdingtheslidesparalleltoeachother.Theslideswereimmersedinbufferandorientedperpendicularlytotheincidentmeasuringbeam.Abaselinereferencewasrecordedbyusingfourblank,cleanedquartzslidespositionedinthesameVisibleAbsorptionSpectroscopyandLinearvisabsorptionspectrawererecordedwithaPerkin-ElmerLambda2UVvisspectrophotometer.Rectan-gularquartzcuvettesof0.2and1.0cmpathlengthswereusedformeasuringsolutionspectra.Forfilmspectra,quartzslidescoatedwithLBfilmwerepositionedinaquartzcuvetteof1.0cmpathlengthusingaTeflonblock(whichfitstightlyonthebottomofthecuvetteandhasoneslitat0,30,45,or60anglerelativetotheedgesoftheblock)holdingtheslideataselectedanglewithrespecttotheincidentmeasuringbeam.Theslideswerealwaysimmersedinbuffer.Forlineardichroismmeasurements,aUVdichroicpolarizerfromOrielCorporation(Stratford,CT)sensitivefrom230to770nmwasplacedbetweentheincidentbeamandthecoatedslide.Inallcasesabaselinereferencewasrecordedbyusingablank,cleanedquartz1.SolutionCharacterizationofHemeThe(proteinmaquettewasfoundtohaveverydifferentsolubilitypropertiesfromthe(proteinmaquettes,evidentlyresultingfromitshighlyasymmetricchargedistribution.Ationicstrengthsupto1M,solutionsarehighlyscatteringinthepHrangefrom4.5(pofglutamate)to10.2(poflysine),asshowninpartsAandBofFigure2.However,solubilitywithinthisrangeincreaseswithincreasingionicstrength(partsCandDofFigure2).Thus,atanionicstrengthof1.5M,theproteincanbereadilysolvatedevenatneutralpH,withoutcausingprecipitation.ThisisdonebyexposingtheproteintopH4.4HemeProteinMaquetteMonolayersJ.Phys.Chem.B,Vol.103,No.42,1999 bufferwith1.5MNaClfollowedbyraisingthepHto8.0(Figure2D).Hemecanbeincorporatedintothemaquetteinthehighionicstrengthsolutionthusprepared.Theapparentmolecularweightsathighionicstrengthdeterminedbygelpermeationchromatographywere17.3and18.2kDabeforeandafterincorporationofhemes,whichareverysimilartothe(andhememaquettes,andconsistentwitha-helix-bundleaggregationstateforboth(.Figure3AshowsthathemehasaCDspectrumtypicalofahighly-helicalstructurewithminimaat208and222nm,andthehelicalcontent(75%)issimilartothoseofhemeandhememaquettes.Figure3Bshowsthathemehasavisspectrumtypicalofbis-histidineligatedferrichemewithaSoretmaximaat412nmand-bandmaximaat5502.CharacterizationoftheMaquettesonAirThepressureareaisothermsof(,and(withouthemearecomparedinFigure4A,andthepressureareaisothermsoftheheme-containing,heme,andhemecomparedinFigure4B.Theisothermsofallthreemaquettessharethecharacteristicsthatwhenthefilmswerecompressedtoapproximately30mN/m,atransitionfromgaseousstatetoliquidexpandedstateofthemonolayeroccurs;beyondthetransitionpoint,thesurfacepressureincreasesonlytoasmallextentperunitdecreaseofsurfacearea.Thesurfaceareapermoleculeatthetransitionpointprovidesameasureofthemolecularareafororientationsfoundatlowsurfacepressure.Inthelowsurfacepressuregaseousstate,atwo-dimensionalversionoftheidealgaslawapplies:isthesurfacepressure,thesurfaceareaperthelimitingareatheBoltzmannconstant,thetemperatureinkelvin,andthenumberof-helicesperindependentparticle.InFigure4D,valuesareplottedforthelow-pressureregion(from1to4mN/m)ofeachisotherm.Theslopesofthefittedlinesgivethevalueof,andthe-interceptsofthelinesgivethevalueof.WecomparethisareawiththedimensionsofasinglehelixderivedfromanNMRstructure,whichhasahelicaldiameterof1213…,forasmallcrosssectionof144170…,andahelicallengthof50…,foralengthwisecrosssectionofabout600…1.(TraceainFigure4Ashowstheisothermof(,andFigure4Drevealsthatitsvalueis2.09,whichindicatesthattwo-helicesareassociatedintheindependentparticlesontheairwaterinterface.Figure4Dalsorevealsthatforthe(filmis593…-helix,whichindicatesthattheaxesofthe-helicesareparalleltotheairinterface.Thus,thefour--helixbundleappearstobedissociatedintoapairofsubunitsthatarelyingflatontheairwaterinterface,presumablywithallpolarsidesincontactwiththewatersurfaceandhydrophobicfacesexposedtotheair.TracebinFigure4Bshowstheisothermofheme,andFigure4Drevealsthatitsvalueis2.05andis602…-helix,indicatingthatthemaquettebothwithandwithouthemeexhibitsthesamebehaviorontheair2.(TracecinFigure4Ashowstheisothermof,andFigure4Drevealsthatitsvalueis4.06,whichindicatesasexpectedthatthefour-helicesareassociatedin Figure2.Solubilityof(atdifferentpHandionicstrength,demonstratedbyUVspectraofMpeptidesolution.Acleantryptophanspectrumwithaflatbaselinenear400nmindicatesgoodsolubility,whileabroadenedtryptophanspectrumwithaslantedbaselineindicatesaggregationinthesolution(alsovisiblebylookingthroughthesolution).PartAshowsthat(wassolublein200mMNaClbufferat4.4(wheremanyglutamatesareneutral)butaggregatedwhenthepHwasadjustedto5.0.PartBshowsthat(wassolublein200mMNaClbufferatpH10.5(wheremanylysinesareneutral)butaggregatedwhenthepHwasadjustedto10.2.PartCshowsthatwassolublein1MNaClbufferatpH4.4,andsomeaggregationwasobservedwhenthepHwasadjustedto8.0.PartDshowsthat(wassolublein1.5MNaClbufferatpH4.4,andnoaggregationwasobservedwhenthepHwasadjustedto Figure3.Characterizationofhemein10mMphosphate,1.5MNaCl,pH8.0buffer.PartAshowsacomparisonofCDspectraofheme(opencircles)andheme(solidline).Forheme,theCDspectropolarimeterwassaturatedatwavelengthsbelow200nmbecauseofthehighabsorptionofUVlightbythehighconcentrationsalt.PartBshowstheUVvisabsorptionspectrumofhemeJ.Phys.Chem.B,Vol.103,No.42,1999Chenetal. theindependentparticlesontheairwaterinterface.Figure4Dalsorevealsthatthefor(is573…-helix,whichalsoapproximatelycoincideswiththeareaofeach-helixiftheyareparalleltotheairwaterinterface.Thus,althoughthe-sunitsarestilllinkedtogetherbythedisulfidebond,theydonotformafullyassociatedbundlebutratherlieflatside-by-sideorend-to-endontheairwaterinterfacewithpolarfacesdownandhydrophobicfacestotheairasdiscussedforTracedinFigure4Bshowstheisothermofheme,andFigure4Drevealsthatitsvalueis3.91andis580…-helix,indicatingthatthemaquettebothwithandwithouthemeexhibitsthesamebehaviorontheair3.(TraceeinFigure4Ashowstheisothermof,andFigure4Drevealsthatitsvalueis3.89,whichindicatesasexpectedthatthefour-helicesareassociatedintheindependentparticlesontheairwaterinterface.How-ever,Figure4Dalsorevealsthatthefor(is322-helix,whichisapproximatelyhalfoftheparallelarea.Thisvaluecoincideswiththeconfigurationinwhichthe-helixbundleliesparalleltotheairwaterinterfacebutwithtwohelicesontopoftheothertwo;italsoindicatesthatthefour--helix-bundlestructureremainsintegralandorientsitshelicalaxesparalleltotheairwaterinterface.TracefinFigure4Bshowstheisothermofheme,andFigure4Drevealsthatitsvalueis4.19,whichindicatesasexpectedthatthefour-helicesareassociatedintheindependentparticlesontheairwaterinterface.Inthiscasevalueforhemeis194…-helixandhencequitedifferentfromallothermaquettes.Thisvalueisclosetotheexpectedcrosssectionareaofan-helixwhenitstandsperpendiculartotheairwaterinterface.Thisanalysisindicatesthatthefour--helix-bundlestructureofhemeremainsintegralandorientsperpendiculartotheairTracesgandhinFigure4Cshowtheisothermsof(palmandhemecharacterizedinourprevi-ouspaper.Thelowsurfacepressureregionoftheisothermsof(palmandhemeisverysimilartothatof(andheme.Figure4Drevealsthatvalueis1.81for(palmand1.83forheme,andthevalueis590…-helixfor(palmand598…-helixforheme.Theseindicatethatatlowsurfacepressurethenumberofhelicesassociatedasindependentparticlesandhelicesorientationofandhemearesimilartothoseof(andhemeRemarkably,uponcompressiontohighsurfacepressure,theisothermsof(palmandhemeanotherphasetransitionthatdiffersgreatlyfromtheisothermsof(andheme.Thisphasetransitionwastocorrespondtothechangeof-helicesorientationfromparalleltoperpendiculartotheairwaterinterface(notethatthishighsurfacepressureregiondoesnotlenditselftoanalysisbyeq1).Thesurfaceareaper-helixatthissecondphasetransitionissimilartothesurfaceareaper-helixofatthefirstphasetransition(tracefinFigure4B).Thismatchaddsweighttotheconclusionthatbothmaquettesorientwith-helixaxesperpendiculartotheairwaterinterface.Thedifferenceisthathighsurfacepressuresarerequiredtolift(palmandhemeoutofthedissociatedsubunitstagewithhorizontalintotheverticalorientation,whilehemetheverticalorientationspontaneously.3.CharacterizationoftheHemeProteinMaquettesonLBFilms.Thehemeproteinmaquettesheme,heme,andhemeweretransferredtoquartzsubstratesandstudiedbyUVvisandCDspectra.UVspectrarevealedthehemeligationstatus,andCDspectrarevealedthe-helicalsecondarystructurecomposition.CD Figure4.Orientationofmaquettesontheairwaterinterfacecharacterizedbysurfacepressureareaisotherms.Thesubphaseis1mMphosphatebuffer(withnosalt),C.PartAshowstheisothermsofthemonolayerfilmsderivedfromthreemaquetteswithoutheme:((a),((c),and((e).PartBshowstheisothermsofthemonolayerfilmsderivedfromthreemaquetteswithheme:heme(b),heme(d),and(f).PartCshowstheisothermsofthemonolayerfilmsderivedfrom(palm(g)andheme(h)(characterizedinref12).PartDshowsplotsoffrom1to4mN/m.Theexperimentalpoints(opencircles)arefittedwithlinearequations)(solidlines):(a)(593…-helix;(b)heme602…-helix;(c)(573…(d)heme580…-helix;(e)(322…-helix;(f)heme194…-helix;(g)(palm590…helix;(h)heme598…OrientationsofthemaquettesontheairwaterinterfacethatareconsistentwiththeobservedvaluesareillustratedneartheisothermsinPartsAHemeProteinMaquetteMonolayersJ.Phys.Chem.B,Vol.103,No.42,1999 spectraalsoindicatedthe-helicalorientations,whileUVvislineardichroismrevealedtheorientationofthehemeplanerelativetothesubstrateplane.DepositionRatio.IntheLBmethod,therelativelyhydro-phobicairsideofthesurfacefilm,shouldadheretoahydrophobicalkylatedsubstrateuponimmersion.However,theair-facingsurfaceisnotnecessarilystronglyhydrophobic,justlesshydrophobicthanthewater-facingfilmsurface;thus,depositionmaynotbeperfect.ThedepositionratioofhemeandhemeontransferfromtheLBtroughtothequartzslideswasfoundtobeclosetounity,whilethedepositionratioofhemewasnotfoundtobesohigh;valuesintherange0.60.8weretypical.VisibleSpectra.TheUVvisspectraofheme,heme,andhemeinsolutionalldisplaythesameabsorptionpropertiescharacteristicofintactbis-histidinehemeligationinboththeferricandferrousforms.ThespectraofhemeandhemeasLBfilmsonquartzalsodisplaythecharacteristicUVvisabsorp-tionpropertiesofbis-histidinehemes(Figure5),andtheyareidenticaltothespectraofthemaquettesinsolution(Figure3B).Hence,itisevidentthatthebis-histidinehemeligationisconservedontheseLBfilms.Incontrast,thespectrumofLBfilmofhemediffersfromtheothersandshowsabsorptionpropertiesofunligatedhemes(Figure5),indicatinglossofhemeligationonthefilm.Thesefilmswerenotanalyzedfurther,andpossiblereasonsforlossofhemewillbediscussedThedensityofthebis-histidineligatedhemesofthehemeandhemeintheLBfilmswasestimatedfromtheabsorbanceoftheSoretbandmaximumbetween411and412nm,assumingthesolutionextinctioncoefficient(120000M)appliesandtakingintoaccountthedepositionratio.Thehemedensityinthehemefilms(depositionratiounity)wasestimatedtobe1230…histidineligatedheme.Thisisequivalentto615…-helixanddirectlyconsistentwiththe602…-helixvaluefoundbytheisothermontheairwaterinterface.ThehemedensityintheLBfilmswasestimatedtobe570…histidineligatedheme,whichaftercorrectionforthedepositionratio(heremeasuredtobe0.7;seeearlier)isequivalentto200-helix,againconsistentwiththe194…-helixvaluedeterminedbytheisotherm.CDSpectra.Figure6comparestheCDspectraofheme,andhemeLBfilmsandtheircorrespondingsolutionspectra.TheCDspectraoftheLBfilmsconservetheshapethatischaracteristicofapredominately-helicalstructure.Wenote,however,thattheratiooftwominimaat208and222nmoftheLBfilmspectradiffersfromthatofthesolutionspectra.TheCDbandat208nmintheand(LBfilmsisconsiderablymorenegativeincomparisontotheirsolutionspectra,whilethe208nmbandinthehemeLBfilmisconsiderablylessnegativeincomparisontoitssolutionspec-TheMoffitexcitontheorypredictsthatthebandat208nmispolarizedparallelto-helices.Thispredictionwasprovedbyseveralexperiments,includingtheworkofOlahandHuangetal.anddeJonghetal.Sincetheincidentlightisnormaltothesubstrate,amorenegative208nmbandcorre-spondstoapredominantlyparallelorientationoftheheliceswithrespecttothesubstratesurfaceandalessnegative208nmbandcorrespondstoapredominantlyperpendicularorienta-tionofthehelices.Thus,thehelicesinhemeLBfilmsarepredominantlyparalleltothesolidsubstratewhilethehelicesinthehemeLBfilmarepredominantlyperpendiculartothesubstrate,resultsentirelyconsistentwiththeirorientationsidentifiedbyLBisothermsontheairwaterinterface.VisibleLinearDichroism.ThedichroicratioofabsorptionofUVvislightlinearlypolarizedperpendicularversusparalleltotheplaneofincidencerevealstheaveragetiltangleofthehemeplanewithrespecttothesubstrate.Itisgenerallyassumedthattheelectronictransitionsresponsiblefor Figure5.visabsorptionspectraofthehemeproteinmaquetteLBfilms.SpectraofLBfilmsderivedfromheme(a),heme(b),andheme(c)arecompared.SpectraweremeasuredwithtwoslidescoatedwithamonolayerLBfilmonbothsides,arrangedperpendicularlytothemeasuringlightbeam. Figure6.CDspectraofthemaquetteLBfilmsandcomparisonwithrespectivesolutionCDspectra:(A)hemeLBfilmspectrum(darkline)andsolutionspectrum(grayline);(B)(LBfilmspectrum(darkline)andsolutionspectrum(grayline);(C)hemeLBfilmspectrum(darkline)andsolutionspectrum(grayline).LBfilmspectraweremeasuredwithfourslidescoatedwithmonolayerLBfilmonbothsides,arrangedperpendicularlytothemeasuringlightbeam.ThescalesofthesolutionspectrawereadjustedtocomparewithLBfilmspectra.J.Phys.Chem.B,Vol.103,No.42,1999Chenetal. theSoretand-bandsinhemesareisotropicallypolarizedintheplaneoftheheme.ArelationshiphasbeendescribedestimatetheaverageorientationofthehemegroupinhemeistheincidentangleoftheincominglightontheLBfilmandistheanglebetweenthenormalofthehemeplaneandthenormalofthesubstrateplane.Figure7showstheUVvisabsorptionspectraofamonolayerLBfilmofhemeandhemeparallelandperpendicularpolarizedincidentlightandwithanincidentangleof60.ThedichroicratioofthehemeLBfilmis0.60,correspondingtoanaverage38tiltangleofthehemeplanerelativethesubstrateplane.Incontrast,thedichroicratioofthehemeLBfilmis1.14,correspondingtoanaverage60tiltangleofthehemeplanerelativethesubstrateplane.Asshowninourpreviouspaper,ifweassumethatthehemeplanesareparalleltothe-helixaxis,anaverage38tiltangleofthehemeplaneimpliesthattheaveragehelixtiltangleissomevaluebetween0and38,andlikewise,anaverage60tiltangleofthehemeplaneimpliesthattheaveragehelixtiltangleisbetween0and60.Thus,wecannotinferthehelixtiltanglefromthesehemeplanetiltangles.Nevertheless,theseresultsfurthersupporttheCDresultsthatindicatedthatthehelicesinhemeLBfilmsarepredominantlyparalleltothesolidsubstrateandthoseinhemeLBfilmsarepredominantlyperpendiculartothesubstrate.Moleculesatairwaterinterfacesareexposedtoverystrongsurfacetensionforcesquitedifferentfromtheforcesinbulkaqueousphase.Theseforcescanhaveaprofoundeffectonthemolecularstructureoffour--helixbundlesoriginallydesignedforstabilityinthebulkaqueousphase.Whilethehydrophobicforcewilldrivebundleassemblyinsolutionbyminimizingthehydrophobicsurfaceareaexposedtosolvent,attheairinterfacethisforcewillbeasymmetricandbundleformationisnotensured.Carefulredesigncancreatemoleculeswithbothdesirablesolubilityinthebulkaqueousphaseandcontrolledself-assemblyattheinterface.Inthecaseoftheoriginal(maquettes(withorwithouthemes),thehydrophobicforcesandspiraldistributionofthechargedresiduesdrivingbundleassociationinsolutionM)doesnotensurebundleassemblyattheairwaterinterface.Thehydrophobicairphaseappearstosuccess-fullycompetewiththehydrophobicinteractionsofthefour-helix-bundlecoretosimplyopenupthehydrophobicinterfacebetweentwounitsandexposethehydrophobicfaceofeachofthetotheair,asshowninFigure8(theshadedregionsinpartsCandDofFigure8representthehydrophobicpartsofthehelices).However,becauseaddedhemebindsinternallyintheunit,thebis-histidineligationsofthehemescanbemaintainedontheairwaterinterface,asshowninpartsBandDofFigure8.Inthecaseof(maquettes(withorwithouthemes),-helix-bundleformationisalsooverwhelmedontheairwaterinterface.However,thetopologicalconstraintsofbundledissociationinthecovalentlylinkedfour-bundlearemuchdifferentthaninthenoncovalentlylinked(.Whilethe(-helixbundlecansimplydissociateattheinterfaceintounitsthatmaintainhistidinesinproximityforhemebinding(Figure8),thecovalentlylinkedbundlecanpartiallydissociateinseveralways,onlysomeofwhichmaintainhistidinesinproximityforhemebinding(Figure9).TheconfigurationshowninpartsAandBofFigure9inprinciplecanmaintainbindingoftwohemes,andtheconfigurationshowninpartsCandDofFigure9inprinciplecanmaintainbindingofonehemebetweenhelices2and3.Itisclearthatnosignificantbindingofhemeoccurs(Figure5).Wesuggestthatinterferencefromhelices1and4forcethehistidinepairofhelices2and3topointawayfromeachother,causingthelossoftheheme,asshowninpartsEandFofFigure9.Anadditionalandpossiblysignificantcontributortohemelossinasurfacereorganizationprocessisthatatransientlossmaybeirreversible.Thedissociationofwater-solublebundlesattheairinterfacecanbeconqueredwithseveralstrategies.Whiletheadditionofhydrophobictailsto(maquettes,suchasmaquettes(withorwithouthemes),didnot Figure7.vislineardichroismofmaquetteLBfilms.PartAshowstheabsorptionspectraofamonolayerhemeLBfilmwithincidentmeasuringlightpolarizedparallel(dashedline)andperpendicular(solidline)totheplaneofincidence.Theincidentangleis60.PartBshowstheabsorptionspectraofamonolayerhemeLBfilmwithparallel(dashedline)andperpendicular(solidline)polarizedincidentmeasuringlight.Theincidentangleis2sin 1+cos2ãsin2õ(2) Figure8.Illustrationsof(andhemetransferredfromaqueoussolutiontotheairwaterinterface.Therhombusesinthisfigureandthefollowingfiguresindicatethehorizontalplaneanddonotrepresenttheexactpositionoftheairwaterinterface.PartsA[(]andB[heme]bothshowthatthefour--helixbundlesdissociateintothesubunitswithhorizontallyorientedheliceswhentransferredtotheairwaterinterface.Thehistidinepairsremaininproximityforhemebinding.PartsC[(]andD[heme]provideaviewalongthehelixaxis,inwhichtheshadedregionsrepresentthehydrophobicpartsofthehelices.HemeProteinMaquetteMonolayersJ.Phys.Chem.B,Vol.103,No.42,1999 preventthefour--helixbundlesfrombeingevertedbythesurfaceforcesatlowsurfacepressure,asshowninpartsAandBofFigure10,athighsurfacepressurethefour--helixbundlesmaybereassembled.Apparently,athighpressurethebundlesareforcedintothewaterphase,likethetail-free(,butanchoredattheinterfacebythehydrophobictailsstickingoutintheairphase,showninpartsCandDofFigure10.The(maquettesrepresentadesignthatcanresisttheevertingforcesattheairwaterinterfaceevenatlowsurfacepressure.Inthisdesign,likechargesweresegregatedontodifferenthelicesofthefour--helixbundlestocreatestrongattractiveforcesbetweenadjacenthelices.Theseexternalinterhelixelectrostaticattractionsaddtothehydrophobicforcesdrivingbundleformationandcreatestablewater-solublemaquetteframesevenatsurfaces.Yetanotherstrategyinvolvestheuseofinternalcofactors.The(maquettewithandwithouthemeswerefoundtohavedifferentorientationsonairwaterinterfaces,asshowninpartsAandBofFigure11.Atthesurfacetherewillbeabalancebetweentwoforces:electrostaticforces,whichwilltendtorotatehelicestomaximizetheattractiveinteractionbetweencomplementarychargedsurfaces,andhydrophobicforces,whichwilltendtominimizetheexposureofthehydrophobicresiduesofthesehelicestotheaqueoussubphase.Introducingthelargelyhydrophobichemecofactormaysig-nificantlyshiftthisbalancetofavorthegeometryofFigure11B,withhelicesorientedmoreperpendiculartothefilmsurface.Inanotherviewofthisbalance,proteinrepackinguponhemebindingmayshiftthedistributionofpartlyexposedhydrophobicresiduesofthecoretofavoranorientationwithoneendofthebundleexposedtotheair.Segregationoflikechargesontodifferenthelicesisinasenseanextremedesignbecausetheattractiveforcesbetweenthehelicesaresostrongthatunderlowionicstrengthconditionstheycausethebundlestoaggregate.However,chargesegrega-tionisacommondesigninnaturewhererelativelylargepatchesoflikechargesaredistributedononesideofaprotein,suchasandplastocyanin.Thiskindofchargedistributionusuallypromotesthebindingofproteinstoanoppositelychargedsurface.Themodificationofthechargedresiduesdistributionindenovodesignedproteinsoffersanengineeringtechniquetocontroltheassemblyofproteinsnotonlyatthewaterinterfacebutonsurfacesingeneral.ThisresearchwassupportedbyagrantfromNIHGM41048andinpartbyanMRSEC/IRGgrantfromNSF(DMR96-20668).ReferencesandNotes(1)DeGrado,W.F.;Wasserman,A.R.;Lear,J.D.(2)Bryson,J.W.;Betz,S.F.;Lu,H.S.;Suich,D.J.;Zhou,H.X.;O'Neil,K.T.;DeGrado,W.F.,935(3)Tuchscherer,G.;Scheibler,L.;Dumy,P.;Mutter,M.,63(4)Moser,C.C.;Keske,J.M.;Warncke,K.;Farid,R.S.;Dutton,P.,796(5)Bendall,D.S.,Ed.ProteinElectronTransfer;BIOSScientificPublishersLtd.:Oxford,1996.(6)Robertson,D.E.;Farid,R.S.;Moser,C.C.;Urbauer,J.L.;Mulholland,S.E.;Pidikiti,R.;Lear,J.D.;Wand,A.J.;DeGrado,W.F.;Dutton,P.L.,425(7)Choma,C.T.;Lear,J.D.;Nelson,M.J.;Dutton,P.L.;Robertson,D.E.;DeGrado,W.F.J.Am.Chem.Soc.,856(8)Rabanal,F.;DeGrado,W.F.;Dutton,P.L.J.Am.Chem.Soc.,473(9)Gibney,B.R.;Rabanal,F.;Reddy,K.S.;Dutton,P.L.,4635(10)Shifman,J.M.;Moser,C.C.;Kalsbeck,W.A.;Bocian,D.F.;Dutton,P.L.,16815 Figure9.Illustrationsof(andhemetransferredfromaqueoussolutiontotheairwaterinterface.PartsAA(R-l-R-s-)2]andB[heme]showadissociationtopologyofthefour--helixbundlesattheairwaterinterfacethatmaintainsproximitybetweenhelices1and4andhelices2and3andhencecouldpermitfullbis-histidineligationofhemes.PartsC[(]andDD2-(R-l-R-s-)2]showanotherdissociationtopologyofthefour-helixbundlesattheairwaterinterface,wherethehistidinepairprovidedbyhelices1and4isnolongerinproximitywhilethepairprovidedbyhelices2and3isstillinproximityforhemebinding.PartsE[(]andF[heme]showthatthehistidinepairofhelices2and3pointsawayfromeachothertoimprovethepackingbetweenhelices1and2andhelices3and4.Bis-histidineligationofhemesisnotpossibleinthistopology.InPartsCF,viewsalongthehelixaxisarealsoprovidedinwhichtheshadedregionsrepresentthehydrophobicpartsofthehelices. Figure10.Illustrationsof(palmandhememaquettestransferredfromaqueoussolutiontotheairinterface.PartsA[(palm]andB[hemeshowthatthefour--helixbundlesdissociateintothewithhorizontallyorientedheliceswhentransferredtotheairinterface,similarto(andhememaquettes.PartsCC(2-R-ss-R)2]andD[heme]showthatathighpressurethebundlesareforcedintothewaterphaseandanchoredattheinterfacebythehydrophobictailsstickingoutintheairphase. Figure11.Illustrationsof(andhememaquettestransferredfromaqueoussolutiontotheairwaterinterface.PartAshowsthat(remainasfour--helixbundlesatthewaterinterface,withthehelixaxisparalleltotheinterface.PartBshowsthathemeremainasfour--helixbundlesatthewaterinterface,withthehelixaxisperpendiculartotheinterface.J.Phys.Chem.B,Vol.103,No.42,1999Chenetal. 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