Investigation of structural properties in biomolecular systems using synchrotron-based spectroscopies [Elektronische Ressource] / von Kurt Kummer

Description

Investigation of structural properties inbiomolecular systems usingsynchrotron-based spectroscopiesDissertationzur Erlangung des akademischen GradesDoctor rerum naturalium(Dr. rer. nat.)vorgelegt der Fakult¨at Mathematik und Naturwissenschaften derTechnischen Universit¨at Dresden vonDipl.-Phys. Kurt Kummergeboren am 27. Dezember 1982 in Schwedt/OderEingereicht am 25. November 2009Die Dissertation wurde in der Zeit von Oktober 2007 bis November 2009am Institut fu¨r Festk¨orperphysik angefertigt.Gutachter: Prof. Dr. Clemens Laubschat (TU Dresden)Prof. Dr. Eckhardt Ru¨hl (FU Berlin)AbstractSolid state approaches to structural properties like diffraction or microscopy tech-niques often cannot be applied to biomolecular systems, at least not without special post-preparation which often corrupts the desired properties of the pristine systems. In thiswork the capabilities of synchrotron-based, soft X-ray spectroscopies as an alternative wayto unravel structural properties of such systems are tested. To this end, three exemplarysystems were investigated each with the focus on another facet and characteristic lengthscale. The first example are DNA-alkanethiol self-assembled monolayers, also known asDNA microarrays or DNA chips, for which a way to monitor and controllably tune thestructural composition onthe mesoscopic scale ofmany thousandsofmolecules wassoughtfor.

Sujets

• Physik

Informations

• Publié par: technische_universitat_dresden
• Publié le: 01 janvier 2009
• Nombre de lectures: 23
• Langue: English
• Poids de l'ouvrage: 14 Mo

Extrait

Investigationofstructuralpropertiesin
biomolecularsystemsusing
synchrotron-basedspectroscopies

Dissertation
zurErlangungdesakademischenGrades
Doctorrerumnaturalium
(Dr.rer.nat.)

vorgelegtderFakulta¨tMathematikundNaturwissenschaftender
TechnischenUniversita¨tDresdenvon

Dipl.-Phys.KurtKummer

geborenam27.Dezember1982inSchwedt/Oder

Eingereichtam25.November2009

DieDissertationwurdeinderZeitvonOktober2007bisNovember2009
amInstitutfu¨rFestko¨rperphysikangefertigt.

Gutachter:

rP.fo

.forP

.rD

.rD

Clemens

Laubschat

Eckhardt

lhu¨R

(UF

UT(

Dresden)

Berlin)

Abstract
Solidstateapproachestostructuralpropertieslikediractionormicroscopytech-
niquesoftencannotbeappliedtobiomolecularsystems,atleastnotwithoutspecialpost-
preparationwhichoftencorruptsthedesiredpropertiesofthepristinesystems.Inthis
workthecapabilitiesofsynchrotron-based,softX-rayspectroscopiesasanalternativeway
tounravelstructuralpropertiesofsuchsystemsaretested.Tothisend,threeexemplary
systemswereinvestigatedeachwiththefocusonanotherfacetandcharacteristiclength
scale.TherstexampleareDNA-alkanethiolself-assembledmonolayers,alsoknownas
DNAmicroarraysorDNAchips,forwhichawaytomonitorandcontrollablytunethe
structuralcompositiononthemesoscopicscaleofmanythousandsofmoleculeswassought
for.Thesecondexamplefocusesonthesingle-moleculeandsubmolecularscaleinmetal-
proteinhybridcompoundswiththeaimtoidentifythebindingsiteofmetalatomsorions
withinproteinmoleculesandtheunderlyinginteractionmechanisms.Themostfundamen-
talstructuralscale,thelevelofsinglebondsandmolecularorbitals,isaddressedinthe
lastexamplewhereitwastriedtoelaborateanapproachtomapthetopologyofmolecular
orbitalsbaseduponX-rayabsorptionproperties.Thisapproachwasputtothepractical
testforthecharacteristic*peptideorbitalsinproteinbackbones.Forallthreeinvestigated
examples,spectroscopiesusingsoftX-raysynchrotronradiationwereabletoextractthe
desiredinformation,thusconrmingthattheymaygrantalternativeaccesstostructural
propertiesofsoft-mattersystemsincaseswherestandardapproachesfail.

Kurzfassung
KlassischeFestko¨rpertechnikenzurStrukturuntersuchung,wieStreu-oderMikrosko-
piemethoden,ko¨nnenha¨ugnichtaufBiomoleku¨lsystemeangewandtwerden,zumindest
nichtohnespeziellePostpra¨paration,diedieurspru¨nglichenEigenschaftendieserSysteme
oftverfa¨lscht.IndieserArbeitsolluntersuchtwerden,inwieweitRo¨ntgenspektroskopien
basierendaufSynchrotronstrahlungeinenalternativenZugangzuStruktureigenschaften
solcherSystemebieten.DazuwurdendreiSystemeexemplarischuntersucht,jeweilsmit
SchwerpunktaufeinenanderenAspektundcharakteristischenLa¨ngenbereich.Fu¨rselbst-
organisierendeDNA-Alkanthiol-Schichten,sogenannteDNA-Chips,wurdenacheineWeg
gesucht,ihrestrukturelleZusammensetzungaufdermesoskopischenEbenevielertausend
Moleku¨lezubestimmenundkontrolliertzumodizieren.MetallisierteProteinstrukturen
wurdenaufEinzelmoleku¨l-bzw.submolekularerEbeneuntersucht,mitdemZiel,dieOrte
derMetallanlagerunginnerhalbdesProteinsunddiezugrundeliegendenWechselwirkungs-
mechanismenzuidentizieren.DieunterstestrukturelleEbene,derBereicheinzelne
BindungenundMoleku¨lorbitale,wurdeadressiertamBeispielder*peptideOrbitaledes
Proteinru¨ckrats.Dafu¨rwurdeeineMethodezurKartographierungeinzelnerOrbitalean-
handvonRo¨ntgenabsorptionseigentschaftenherausgearbeitetundpraktischgetestet.In
allendreiFa¨llenkonntenRo¨ntgenspektroskopiendieno¨tigenInformationenliefernund
damitihrPotentialfu¨rStrukturuntersuchungeninweicherMaterieunterBeweisstellen.

Contents

1Preface

3

2Synchrotron-basedspectroscopiesinthesoftX-rayrange5
2.1Photoelectronspectroscopy..........................5
2.1.1Basicprinciples.............................5
2.1.2Core-levelenergyshiftsinphotoemissionspectra...........7
2.1.3Analysisofcore-levelPEspectra...................10
2.1.4Instrumentation.............................12
2.2X-rayabsorptionspectroscopy.........................15
2.2.1Crosssectionandoscillatorstrength.................15
2.2.2CharacteristicsofX-rayabsorptionspectra..............17
2.2.3DetectionofX-rayabsorptionsignals.................25

3MonitoringandtuningstructuralpropertiesinDNAmicroarrays:Alook
intoself-assembledstructuresofbiomoleculesonthemesoscopicscale29
3.1Introduction...................................29
3.2XPScharacterizationofmercaptohexanolself-assembledmonolayers...31
3.3Core-levelelectronicstructureofDNA....................35
3.4TuningtheprobedensityinmixedDNA-MCHmonolayers.........40
3.5InuenceofthesolventtypeontheDNA-SAM...............45
3.6CompetitiveinterplaybetweenMCHandthiolizedDNA..........47
3.7Summary....................................50

4Modicationofthepeptidebondbyatomicandioniccalcium:Metal
bindingmechanisminproteinstructures51
4.1Introduction...................................51
4.2Bindingsiteofcalciumintheproteinstructure...............53
4.2.1Experimentalresults..........................53
4.2.2Abinitiocalculations..........................59
4.3Evidenceforcalciumpenetrationintotheproteinlayer...........61
4.4Chemicalkineticsofthemetalbindingreaction...............63
4.5Conclusions...................................67

1

5Oscillatorstrengthofthe1s!*X-rayabsorptionresonancesofthe
peptidebond:Anapproachtomappingofindividualmolecularorbitals69
5.1Introduction...................................69
5.2Characterizationandrenementofthebeamlinephotonux........70
5.2.1Beammonochromatization.......................70
5.2.2Photonux...............................73
5.3SpectraldependenceoftheX-rayabsorptionofproteins...........75
5.4Finestructureofthe1sX-rayabsorptionedgesinproteins.........77
5.4.1N1sabsorptionedge..........................77
5.4.2C1sabsorptionedge..........................79
5.4.3O1sabsorptionedge..........................80
5.5AbsoluteX-rayabsorptioncrosssectionsandoscillatorstrengths......82
5.5.1Normalizationtotheabsolutescale..................82
5.5.2Oscillatorstrengthofthepeptidebondresonances..........84
5.6Correlationbetweenoscillatorstrengthsandtheorbitaltopology......87
5.7Conclusions...................................89
6Summary91
Bibliography93
AExperimentaldetails105
A.1Beamlinesandexperimentalend-stations...................105
A.1.1Russian-GermanbeamlineatBESSYII................105
A.1.2BeamlineD1011attheMAX-labfacility...............106
A.2Samplepreparation...............................106
A.2.1Mercaptohexanolself-assembledmonolayers.............106
A.2.2GenomicDNA.............................107
A.2.3DierentMgCl2concentrations....................107
A.2.4Dierenttypesofsolvent........................107
A.2.5SimultaneousDNAimmobilizationandSAMformation.......108
A.2.6BacterialsurfaceproteinlayersonSiOx/Si..............108
Acknowledgments111
Eigensta¨ndigkeitserkla¨rung113

1

Preface

Sinceabout20yearsagowhenbiotechnologyandorganicsemiconductortechnologiesbegan
toriseresearchintoorganicandbiomoleculeshasfoundarenaissanceandhasconstantly
intensiedeversince.Todaythesesystemsaresubjecttooneofthemostlivelybranchesof
materialscienceandtheinexhaustiblefundoforganiccompoundsmakesthemanoceanic
poolfordiversetechnologicalapplicationsspanningvariousimaginableeldsfromorganic
solarcellsandLEDsoverbiosensorstomolecularcomputing.Themainreasonforthis
successisthatthesenewmaterialsdorevealnumerousnewanduniquepropertieswhen
comparedtohithertoemployedinorganiccompounds.
Oneandpossiblythemoststrikingprincipallynewaspectoforganicandbiological
systemsistheircapabilitytoself-organizeintoevenhighlycomplexfunctionalstructures.
Withthatbottom-upfabrication,i.e.theself-assemblyofentiretechnologicalunitsfrom
soluteswithoutexternalsteering,comesintosight.Naturally,thedevelopmentandpur-
posefulapplicationofsuchrevolutionarynewfabricationmethodswouldalwaysrequirea
waytomonitorcompositionandpropertiesoftheresultingnanoscopicstructures.Unfor-
tunately,thosetechniquesthatareroutinelyappliedtocharacterizestructuralproperties
of”classical”,inorganicsystemsareinmanycasesnotwell-suitedforthispurpose.Re-
ciprocalorreal-spaceimaging,i.e.diractionorhigh-resolutionmicroscopytechniques,
reachtheirlimitationswhenitcomestosoftmatterstructures.Microscopicstudiesare
oftennotsuitable,becausetheyarerelativelyslow,notchemicallyselectiveandrequire
specialpost-preparationofthesamples,whichrulesthemoutforinsitumonitoringofthe
formationprocessofsuchstructures.Theabsenceof3-dimensionalcrystallineorder,on
theotherhand,oftenprecludestheuseofdiractionmethods.1
Incertaincasestheapplicationofsynchrotron-basedspectroscopiescouldopenan
alternativepathwaytowardsthedesiredstructuralinformation.Althoughthesespectro-
scopiesaddresstheelectronicratherthanthespatialstructureofmattertheycanalso
providewithsomeinsightintothelatterbecauseoftheclosecorrelationbetweenboth.In
thepresentwork,thecapabilityofspectroscopiesusingsoftX-raysynchrotronradiation
forstructuralcharacterizationofbiomolecularsystemsistestedatthreedistinctexam-
1Notethat,todate,X-raydiractionisthemajortooltoexplorethespatialstructureofproteins.
However,itsapplicationisboundtotheavailabilityofspeci