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Development and characterization of a unique photoactivatable label [Elektronische Ressource] / Susan Gayda. Betreuer: G. U. Nienhaus

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167 pages
Ajouté le : 01 janvier 2011
Lecture(s) : 31
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Development and Characterization of
a Unique Photoactivatable Label
Zur Erlangung des akademischen Grades eines
DOKTORS DER NATURWISSENSCHAFTEN
(Dr. rer. nat.)
Fakult¨at fur¨ Chemie und Biowissenschaften
Karlsruher Institut fur¨ Technologie (KIT) - Universit¨atsbereich
genehmigte
DISSERTATION
von
Dipl.-Biochem. Susan Gayda
aus
Leipzig
Juni 2011Dekan: Prof. D. Stefan Br¨ase
Referent: Prof. Dr. Gerd Ulrich Nienhaus
Korreferent: Prof. Dr. Tilman Lamparter
Tag der mundlic¨ hen Prufung:¨ 13.07.2011Contents
List of Figures VII
List of Symbols IX
Introduction 1
Relevance of Photoactivatable Fluorescent Proteins . ............ 1
Thesis Outline ................................. 2
I Scientific Background 3
1 GFP-like Fluorescent Proteins 5
1.1 Properties of GFP-like Fluorescent Proteins .............. 6
1.1.1 Three-Dimensional Structure . . . . .............. 6
1.1.2 Formation of the ’GFP’-Chromophore, p-HBI ......... 7
1.1.3 Modifications of the p-HBI Chromophore . ........... 8
1.1.4 Diversity Among GFP-like Proteins . .............. 9
1.2 Photophysical Properties of GFP-like proteins 10
1.2.1 Spectroscopic Properties of the Chromophore ......... 10
1.2.2 pHSensitivityoftheChromophore ............... 11
1.2.3 Excited State Proton Transfer .................. 12
1.2.4 Blinking .............................. 13
1.2.5 ShiftinEmissionColor...................... 15
1.3 Photoactivatable Fluorescent Proteins 15
1.3.1 Irreversible Photoconversion . 16
1.3.2 Reversible Photoactivation - Photoswitching .......... 22
1.3.3 PAFPs Showing Reversible AND Irreversible Photoactivation 25
1.4 FPs in Superresolution Live-Cell Imaging................ 25
III Materials and Methods 29
2 Molecular Biology 31
2.1 Mutagenesis ................................ 31
2.1.1 Site-Directed Mutagenesis . . .................. 31
2.1.2 Random Mutagenesis — Error PronePCR........... 33
2.2 Protein Expression and Purification................... 35
2.2.1 CellCulturing........................... 35
2.2.2 Protein Purification........................ 35
2.2.3 Analytical Gel Filtration..................... 36
3 Tracking Structural Modifications by Spectroscopy 39
3.1 Principles of Light-Matter Interaction .................. 39
3.2 Absorption Spectroscopy . . . . . . . . . . . . ............. 42
3.3 CircularDichroism ............................ 45
3.4 Fluorescence................................ 45
III Results and Discussion 51
4 Combining Reversible and Irreversible Switching Modes - IrisFP 53
4.1 Introduction 53
4.1.1 Structural Background of Photoinduced Color Changes .... 54
4.2 Spectroscopic Characterization of IrisFP . . . . . ........... 57
4.3 Photoactivation in IrisFP ........................ 62
4.3.1 General Aspects of Photoswitching ............... 62
4.3.2 Green-to-Red Photoconversion . . . 66
4.3.3 ModulationofIrreversibleandReversiblePhotoactivationModes 68
4.4 Summary and Conclusions 72
5 A Genetic Label with Multiple Photoactivation Modes – mIrisFP 75
5.1 Introduction................................ 75
5.2 Semi-rational Engineering of mIrisFP . . . . . ............. 76
5.2.1 MolecularEngineering ...................... 76
5.3 Photophysical Characterization of mIrisFP . . . ............ 80
5.3.1 The Thermally Relaxed Ground State . . . . . ........ 81
5.3.2 Photoactivation .......................... 82
5.4 Applications of mIrisFP ......................... 85
II5.4.1 mIrisFP as a Genetically Encoded Marker ........... 85
5.4.2 CombiningPulse-ChaseExperimentsandSuperresolutionImag-
ing ................................. 85
5.5 Summary and Conclusion . ....................... 90
6 Investigation of Photoswitching Using mIrisGFP 91
6.1 mIrisGFP – a Model System 91
6.2 Spectroscopic Characterization of mIrisGFP . . . . . . . . . ..... 91
6.3 EffectofpHontheThermalEquilibriumofthemIrisGFPChromophore 93
6.3.1 Protonation Equilibrium of the CisChromophore ....... 93
6.3.2 of the TransChromophore...... 98
6.3.3 StructuralInterpretationoftheProtonationEquilibriumShift
upon Cis-Trans Isomerization of the Chromophore ......100
6.4 Chromophore Interconversion in mIrisGFP at the Macroscopic Level . 100
6.4.1 Wavelength Dependence of Photoswitching...........100
6.4.2 ExcitingtheAnionicChromophore ...............102
6.4.3 Thermal Recovery of the CisChromophore107
6.4.4 Excitation of the Neutral Chromophore.............110
6.5 EnergeticInterpretation: InterconversionoftheChromophoreSpecies
on the Microscopic Level .........................114
6.5.1 Exciting the Anionic Cis Chromophore - Along the ’Green
Pathway’ . ............................115
6.5.2 Thermal Recovery of the Cis Chromophore - Along the ’Red
Pathway’ .118
6.5.3 ExcitingtheNeutralCis Chromophore-Alongthe’BluePath-
way’ . ...............................119
6.5.4 Exciting the Neutral Trans Chromophore - Along the ’Gray
Pathway’ .............................120
6.6 Summary and Conclusions . . . . . . . . ................121
Summary 123
Zusammenfassung................................126
Bibliography 145
Appendix i
Danksagung................................... i
List of Publications............................... iii
IIICurriculum Vitae ................................ vii
IVList of Figures
1.1 Structure of GFP-like Proteins. . . . .................. 7
1.2 Formation of the p-HBI Chromophore. . . . .............. 8
1.3 Variations of the Chromophore. . .................... 9
1.4 pH sensitivity of EGFP. ......................... 11
1.5 Model of chromophore protonation energetically coupled to an adja-
cent residue. . ............................... 12
1.6 Excited State Proton Transfer ...................... 13
1.7 Single molecule fluorescence trajectories of GFP S65T. . . . ..... 14
1.8 Green-to-red photoconversion of GFP. ................. 15
1.9 Photoactivation in PA-GFP. ....................... 17
1.10 Photoactivation in PAmCherry. ..................... 18
1.11 Photoconversion in EosFP......................... 20
1.12 Natural occurrence and spectral properties of EosFP. . . . ...... 21
1.13 Photoconversion in EosFP. 21
1.14 Reversible photoswitching in Dronpa. . ................. 22
1.15 PhotoswitchingofDronpavariantsfeaturingapositiveandanegative
switching mode. . . . . .......................... 24
1.16 Schematic illustration of the principle of PALM. ............ 27
2.1 Schematic of site-directed mutagenesis............ 32
2.2 Principle of the generation of random clones............... 33
2.3 Steps of protein purification by affinity chromatography utilizing a
TALON matrix. . . ........................... 36
3.1 Jablonski diagram. ............................ 40
3.2 Schematic illustration of the Frank-Condon principle. . ........ 41
3.3 Absorption of light passing through a sample. . . . . . . . . . .... 42
V3.4 Optical setup for the photoswitching experiments mounted at the
Fluorolog II fluorometer. ......................... 47
3.5 Time traces of the emission intensity during photoswitching. . . . . . 47
4.1 IrisFP protein solution after different illumination conditions. .... 54
4.2 IrisFP crystals under different illumination conditions. ........ 54
4.3 Structural differences between EosFP and IrisFP in the chromophore
vicinity. . . ................................ 55
4.4 Structural changes in the chromophore vicinity of IrisFP upon pho-
toswitching. . . . . . . . . . ....................... 57
4.5 Spectroscopic comparison of EosFP and IrisFP. ............ 59
4.6 pH-dependence of thermally relaxed IrisFP. . . 60
4.7 Spectral changes of IrisFP upon pH variation. ............. 61
4.8 Photoinduced transformations in IrisFP. ................ 62
4.9 Spectroscopic changes upon photoactivation. .............. 63
4.10 ThermalrecoveryofIrisFP. . ...................... 64
4.11 Time traces of IrisFP photoactivation. . . . 65
4.12 Photoswitching cycles of IrisFP. . .................... 66
4.13 Photoconversion in IrisFP and its side effects. . . . .......... 67
4.14 Action spectra of IrisFP. ......................... 69
4.15 Schematicillustrationofthephotoreactionsduring390nm-illumination. 71
5.1 StructuralchangesinmIrisFP....................... 76
5.2 Amino acid sequence alignment of several monomeric anthozoa FPs
andmIrisFP. ............................... 78
5.3 Oligomerization of mIrisFP and its predecessors............. 79
5.4 Absorption and fluorescence spectra of mIrisFP and IrisFP....... 81
5.5 pH-dependence of the thermally relaxed. . . ......... 82
5.6 Time traces of photoactivation and thermal recovery in mIrisFP. . . . 83
5.7 Spectral changes upon photoactivation. . . ............... 84
5.8 Combined pulse-chase experiments and superresolution imaging uti-
lizingmIrisFP. 87
5.9 Focal adhesion dynamics in a live HeLa cell expressing paxillin-mIrisFP. 89
6.1 Spectral properties of mIrisGFP...................... 93
6.2 pH dependence of optical spectra of mIrisGFP in thermal equilibrium. 94
6.3 pH-dep of optical band intensities and positions of the ther-
mally relaxed mIrisGFP chromophore. ................. 95
VI6.4 SchematicillustrationoftheprotonationequilibriumofthemIrisGFP
chromophore interacting with adjacent amino acid side chains. .... 96
6.5 Schematic illustration of the hydrogen bonding network below the
chromophoreuponpHchange....................... 97
6.6 Shift of mIrisGFP Glu212Gln absorption bands. . . .......... 98
6.7 pH-dependent optical spectra of the mIrisGFP chromophore in the
trans state. ................................ 99
6.8 Shift of the transchromophorebandswithpH.............. 99
6.9 Eight-state model comprising the possible chromophore species of
mIrisGFP. .................................101
6.10 Wavelength dependence of the off switching in mIrisGFP. . ......102
6.11 Off switching by excitation within the neutral chromophore band. . . 103
6.12 UV/visible spectra of mIrisGFP, pH 6, before and after illumination
with 473-nm light. . ...........................104
6.13 pH dependence of the off switching quantum efficiency, φ .......105off
6.14 Off switching by different excitation powers. . . ............106
6.15 Absorption spectra collected during thermal recovery of mIrisGFP at
pH7,310K.................................108
6.16 pH dependence of the trans-to-cis isomerization. . ...........109
6.17 Structural rearrangements around the trans chromophore upon pH
changes. . .................................110
6.18 Spectral changes upon off switching with 405-nm light. ........112
6.19 Time traces of the decrease in emission intensity during off switching
with 405-nm light. ............................113
6.20 Isomerization/protonation pathways in mIrisGFP from the energetic
pointofview. ...............................115
6.21 Temperature sensitivity of the off switching. ..............116
6.22 Temp dependence of the mIrisGFP emission spectrum. . . . . 117
6.23 Autocorrelation curve of mIrisGFP. ...................118
6.24 Off switching in the presence and absence of molecular oxygen.....119
VIIVIII