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Pigment pigment interactions and protein dynamics in light harvesting complexes [Elektronische Ressource] : a single molecule study / von Clemens Hofmann

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178 pages
Pigment-PigmentInteractionsandProteinDynamicsinLight-HarvestingComplexes:aSingle-MoleculeStudyVonderUniversita¨tBayreuthzurErlangungdesGradeseinesDoktorsderNaturwissenschaften(Dr.rer.nat.)genehmigteAbhandlungvonClemensHofmannausHamburg1.Gutachter: Prof.Dr.Ju¨rgenKo¨hler2.Gutachter: Prof.Dr. MarkusSchwoererTagderEinreichung: 12.Dezember2003TagdesKolloquiums: 24.Mai2004I certify that all material in this thesis which is not my own work has been identified and thatnomaterial isincludedforwhichadegreehaspreviouslybeenconferred uponme.............................(ClemensHofmann)Scientificpublicationsofresultspresentedinthiswork• CLEMENS HOFMANN, THIJS J. AARTSMA, HARTMUT MICHEL, AND JU¨RGEN KO¨HLER,“SpectralDynamicsintheB800bandofLH2fromRhodospirillummolischianum: ASingle-MoleculeStudy”. NewJour.Phys. 68(2004).¨ ¨• CLEMENS HOFMANN, THIJS J. AARTSMA, HARTMUT MICHEL, AND JURGEN KOHLER,“DirectObservationofTiersintheEnergyLandscapeofaChromoprotein: ASingle-MoleculeStudy”. Proc.Nat.Acad.Sci.U.S.A.10015534-15538(2004).• CLEMENS HOFMANN, FRANCESCOFRANCIA, GIOVANNI VENTUROLI,DIETER¨ ¨OESTERHELT, AND JURGEN KOHLER,“Energytransferinasingleself-aggregatedphotosyntheticunit”. FEBSLett. 546345-348(2003).• CLEMENS HOFMANN, MATIJN KETELAARS,MICHIO MATSUSHITA,HARTMUT MICHEL,¨ ¨THIJS J. AARTSMA, AND JURGEN KOHLER,“Single-MoleculestudyoftheElectronicCouplingsinaCircularArrayofMolecules: Light-Harvesting2ComplexfromRhodospirillummolischianum”. Phys.Rev.Lett.
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Pigment-PigmentInteractionsandProtein
DynamicsinLight-HarvestingComplexes:
aSingle-MoleculeStudy
VonderUniversita¨tBayreuth
zurErlangungdesGradeseines
DoktorsderNaturwissenschaften(Dr.rer.nat.)
genehmigteAbhandlung
von
ClemensHofmann
ausHamburg
1.Gutachter: Prof.Dr.Ju¨rgenKo¨hler
2.Gutachter: Prof.Dr. MarkusSchwoerer
TagderEinreichung: 12.Dezember2003
TagdesKolloquiums: 24.Mai2004I certify that all material in this thesis which is not my own work has been identified and that
nomaterial isincludedforwhichadegreehaspreviouslybeenconferred uponme.
............................
(ClemensHofmann)Scientificpublicationsofresultspresentedinthiswork
• CLEMENS HOFMANN, THIJS J. AARTSMA, HARTMUT MICHEL, AND JU¨RGEN KO¨HLER,
“SpectralDynamicsintheB800bandofLH2fromRhodospirillummolischianum: A
Single-MoleculeStudy”. NewJour.Phys. 68(2004).
¨ ¨• CLEMENS HOFMANN, THIJS J. AARTSMA, HARTMUT MICHEL, AND JURGEN KOHLER,
“DirectObservationofTiersintheEnergyLandscapeofaChromoprotein: ASingle-Molecule
Study”. Proc.Nat.Acad.Sci.U.S.A.10015534-15538(2004).
• CLEMENS HOFMANN, FRANCESCOFRANCIA, GIOVANNI VENTUROLI,DIETER
¨ ¨OESTERHELT, AND JURGEN KOHLER,“Energytransferinasingleself-aggregated
photosyntheticunit”. FEBSLett. 546345-348(2003).
• CLEMENS HOFMANN, MATIJN KETELAARS,MICHIO MATSUSHITA,HARTMUT MICHEL,
¨ ¨THIJS J. AARTSMA, AND JURGEN KOHLER,“Single-MoleculestudyoftheElectronic
CouplingsinaCircularArrayofMolecules: Light-Harvesting2ComplexfromRhodospirillum
molischianum”. Phys.Rev.Lett. 90013004(2003).
¨ ¨• MATIJN KETELAARS,CLEMENS HOFMANN, JURGEN KOHLER, TINA D. HOWARD,
RICHARD J. COGDELL, JAN SCHMIDT, AND THIJS J. AARTSMA,“SpectroscopyonIndividual
Light-Harvesting1ComplexesofRhodopseudomonasacidophila”. Biophys.J.831701-15
(2002).
¨ ¨• JURGEN KOHLER, ANTOINE M. VAN OIJEN, MARTIJN KETELAARS,CLEMENS HOFMANN,
MICHIO MATSUSHITA,THIJS J. AARTSMA, AND JAN SCHMIDT,“OpticalSpectroscopyof
IndividualPhotosyntheticPigmentProteinsComplexes”Int. J.Mod. Phys. B153633-3636
(2001).
Contributionstointernationalconferences
¨ ¨• CLEMENS HOFMANN, THIJS J. AARTSMA, HARTMUT MICHEL, AND JURGEN KOHLER,
“Opticalspectroscopyonindividualphotosyntheticmembranecomplexesfrompurplebacteria”.
InternationalSymposiumonelucidatingbiomolecularnetworksbysingle-moleculetechnologies
inAscona(CH),October2003.
• CLEMENS HOFMANN, MATIJN KETELAARS,MICHIO MATSUSHITA,HARTMUT MICHEL,
¨ ¨THIJS J. AARTSMA, AND JURGEN KOHLER,“SpectroscopyontheB800BandofIndividual
LightHarvesting2ComplexesfromRhodospirillummolischianum”. EURESCOConferenceon
BionanotechnologyinGranada(E),July2003.
• CLEMENS HOFMANN, MARTIN RICHTER, FRANCESCOFRANCIA, DIETER OESTERHELT,
¨ ¨AND JURGEN KOHLER,“BacterialPhotosynthesisinpurplebacteria,PufXandLH1in
Rhodobactersphaeroides: apreliminarystudy”. Springmeetingofthe“DeutschePhysikalische
Gesellschaft”inDresden(D),March2003.
• CLEMENS HOFMANN, MATIJN KETELAARS,MICHIO MATSUSHITA,THIJS J. AARTSMA,
¨ ¨FRANCESCOFRANCIA, DIETER OESTERHELT, AND JURGEN KOHLER,“OpticalSpectroscopy
ofIndividualPigmentProteinComplexes”. VolkswagenStiftung: 3rdinternationalSymposium
onPhysics,ChemistryandBiologywithSingleMoleculesinTutzing(D),September2002.
iii Contributions to international conferences
• CLEMENS HOFMANN, MATIJN KETELAARS,MICHIO MATSUSHITA,THIJS J. AARTSMA,
HARTMUT MICHEL, AND JU¨RGEN KO¨HLER,“SpectroscopyoftheB800BandofIndividual
LightHarvesting2(LH2)ComplexesfromRhodospirillummolischianum”. Springschoolon
SingleMoleculesinPhysics,ChemistryandBiologyinHofgeismar(D),April2002.
• CLEMENS HOFMANN, MATIJN KETELAARS,MICHIO MATSUSHITA,THIJS J. AARTSMA,
¨ ¨HARTMUT MICHEL, AND JURGENKOHLER,“SpectroscopyofIndividualLH2Complexesfrom
Rhodospirillummolischianum —theelectroniccouplingofBChla intheB800ring”. Spring
meetingofthe“DeutschePhysikalischeGesellschaft”inRegensburg(D),March2002.
• CLEMENS HOFMANN, MATIJN KETELAARS,MICHIO MATSUSHITA,THIJS J. AARTSMA,
¨ ¨JAN SCHMIDT, HARTMUT MICHEL, AND JURGEN KOHLER,“Dynamicandstaticbehaviourof
theB800bandofRhodospirillummolischianum”. InternationalWorkshop: MolecularMotorsin
Frankfurt(D),November2001.
• CLEMENS HOFMANN, MATIJN KETELAARS,MICHIO MATSUSHITA,THIJS J. AARTSMA,
¨ ¨JAN SCHMIDT,HARTMUT MICHEL, AND JURGEN KOHLER,“SinglemoleculeSpectroscopyon
individuallightharvesting2complexesfromRhodospirillummolischianum”. Springschool:
OpticalSpectroscopyandMicroscopyofSingleObjectsinLesHouches(F),May2001.
• CLEMENS HOFMANN, MATIJN KETELAARS,MICHIO MATSUSHITA,THIJS J. AARTSMA,
¨ ¨JAN SCHMIDT,HARTMUT MICHEL, AND JURGEN KOHLER,“SinglemoleculeSpectroscopyon
individuallightharvesting2complexesfromRhodospirillummolischianum”. SFB533:
Light-induceddynamicsinbiopolymersinFreising(D),April2001.
• CLEMENS HOFMANN, MATIJN KETELAARS,MICHIO MATSUSHITA,THIJS J. AARTSMA,
¨ ¨JAN SCHMIDT,HARTMUT MICHEL, AND JURGEN KOHLER,“SinglemoleculeSpectroscopyon
individuallightharvesting2complexesfromRhodospirillummolischianum”.
VolkswagenStiftung: 2ndinternationalSymposiumonPhysics,ChemistryandBiologywith
SingleMoleculesinStaffelstein(D),March2001.fu¨r TanjaContents
1 Introduction 1
2 Lightharvestingcomplexes 5
2.1 Photosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
2.1.1 Buildingblocksofthephotosyntheticapparatusinpurplebacteria . . . 6
Pyrrolesandchlorophylls . . . . . . . . . . . . . . . . . . . . . . . . 6
Carotenoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Thereactioncentreandtheantennacomplexes . . . . . . . . . . . . . 8
2.1.2 Thephotosyntheticunit(PSU) . . . . . . . . . . . . . . . . . . . . . . 10
Theperipherallight-harvesting2(LH2)complex . . . . . . . . . . . . 10
Thelight-harvesting1(LH1)complex . . . . . . . . . . . . . . . . . . 12
Thereactioncentre(RC) . . . . . . . . . . . . . . . . . . . . . . . . . 13
Cyclicelectron transportinsideandoutsidethePSU . . . . . . . . . . 13
2.2 Spectral propertiesofpigments . . . . . . . . . . . . . . . . . . . . . . . . . . 14
2.2.1 Theintrinsicelectronicpropertiesofchlorophylls . . . . . . . . . . . . 15
Absorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Emission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
2.2.2 Pigment-proteininteraction . . . . . . . . . . . . . . . . . . . . . . . 17
Thehomogeneouslinewidth,zero-phononlineandphononside-band . 17
Inhomogeneousbroadeningandspectraldiffusion . . . . . . . . . . . 19
2.2.3 Pigment-pigmentinteraction . . . . . . . . . . . . . . . . . . . . . . . 21
Hamiltonoperator: disorderandtypesofinteraction . . . . . . . . . . 22
Pigment-pigmentinteraction, adimermodel. . . . . . . . . . . . . . . 23
Weakinteraction,localised excitations . . . . . . . . . . . . . . . . . . 24
Stronginteraction, Frenkelexcitons . . . . . . . . . . . . . . . . . . . 25
EigenstatesoftheB850pigmentpool . . . . . . . . . . . . . . . . . . 25
3 Materialsandmethods 27
3.1 Linenarrowingtechniquesatlowtemperatures . . . . . . . . . . . . . . . . . 27
3.1.1 Spectral hole-burning . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
3.1.2 Photon-echomeasurements . . . . . . . . . . . . . . . . . . . . . . . 28
3.1.3 Single-moleculespectroscopy . . . . . . . . . . . . . . . . . . . . . . 29
3.2 Thisthesis: singlemoleculespectroscopy . . . . . . . . . . . . . . . . . . . . 29
3.2.1 Prerequisitesforsinglemoleculespectroscopy . . . . . . . . . . . . . 30
3.2.2 Techniquesinlowtemperaturesinglemoleculespectroscopy . . . . . . 31
vvi Contents
Ashorthistory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Confocalmicroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
3.3 Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
3.3.1 Samplepreparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
3.3.2 Listofsamples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
3.3.3 Listofchemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
3.3.4 Atomiccoordinates . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
3.4 Experimentalsetup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Widefieldfluorescencemicroscopy . . . . . . . . . . . . . . . . . . . 35
Confocalfluorescence-excitation spectroscopy . . . . . . . . . . . . . 36
Widefieldfluorescence-excitation spectroscopy . . . . . . . . . . . . . 37
Polarisationdependentspectroscopy . . . . . . . . . . . . . . . . . . . 38
4 TheB800bandofLH2fromRhodospirillummolischianum 39
4.1 StructureoftheB800ring . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
4.2 Experimentalresults . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
4.2.1 Inter-andintra-complexheterogeneity . . . . . . . . . . . . . . . . . . 42
4.2.2 Spectraldiffusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
Spectralswitchingofindividualabsorptionlines . . . . . . . . . . . . 43
Spectraldiffusioncontributingtothehomogeneouslinewidth . . . . . 46
Simultaneousspectral diffusionofabsorptionlines . . . . . . . . . . . 49
Powerdependenceofspectraldiffusion . . . . . . . . . . . . . . . . . 51
4.2.3 Electroniccouplingsinacircular aggregateofmolecules . . . . . . . . 52
Mutualanglesbetweenabsorption-dipolemoments . . . . . . . . . . . 53
Simultaneoustemporal fluctuations . . . . . . . . . . . . . . . . . . . 54
4.3 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
4.3.1 Inter-andintra-complexheterogeneity . . . . . . . . . . . . . . . . . . 55
Statistical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
Comparisonbetweenexperiment andanalysis . . . . . . . . . . . . . . 58
4.3.2 Directobservationofdistincttiersintheenergylandscapeofaprotein . 59
Spectralswitching: Chromophoresprobetheenergylandscape . . . . . 59
Simultaneousspectral diffusion: couplingofchromophores . . . . . . 62
StructuralchangesinthebindingpocketoftheBChla molecules . . . 63
4.3.3 Electroniccouplingsinacircular aggregateofmolecules . . . . . . . . 65
Couplingbetweenneighbouringchromophores . . . . . . . . . . . . . 65
Temporalfluctuationsofthecouplingbetweentwochromophores . . . 67
4.4 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
5 LineshapesoftheB800absorptions 73
5.1 Multivariate statistical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . 73
5.1.1 TheMSAalgorithm . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
5.1.2 UsingMSAinspectroscopy . . . . . . . . . . . . . . . . . . . . . . . 75
5.2 Patternrecognitionontwoexamplespectra . . . . . . . . . . . . . . . . . . . 76
5.2.1 Applicationtosimultaneousspectraldiffusion . . . . . . . . . . . . . . 76
Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
5.2.2 Applicationtospectral switching. . . . . . . . . . . . . . . . . . . . . 78
Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78