Spectroscopic studies and applications of novel fluorescent proteins [Elektronische Ressource] / vorgelegt von Sergey Ivanchenko

Sergey Ivanchenko

Sujets

• Biologie

Informations

• Publié par: universitat_ulm
• Publié le: 01 janvier 2006
• Nombre de lectures: 11
• Langue: English
• Poids de l'ouvrage: 17 Mo

Extrait

Spectroscopic Studies and Applications of
Novel Fluorescent Proteins
Dissertation
zur Erlangung des akademischen Grades Dr. rer. nat.
der Fakult¨at fu¨r Naturwissenschaften
der Universitat Ulm¨
Vorgelegt von
Dipl.-Phys. Sergey Ivanchenko
aus Moskau/Russland
Ulm, 2006Contents
List of Figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV
List of Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IX
Introduction 1
Why Fluorescent Proteins? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Thesis Outline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
I Theoretical Basis 5
1 Introduction to Fluorescent Proteins 7
1.1 Green Fluorescent Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
1.2 Red Fluorescent Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
1.3 Brightness, Stability and Oligomerization of RFPs . . . . . . . . . . . . . . 13
1.4 Optical Highlighters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
1.5 Molecular Basis of Emission Fluctuations of Fluorescent Proteins . . . . . . 16
2 Principles of Absorption and Fluorescence Spectroscopy 19
2.1 The Golden Rule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
2.2 Mechanisms of Spectral Broadening . . . . . . . . . . . . . . . . . . . . . . 20
2.3 Solvent Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
2.4 Two-Photon Excitation Process . . . . . . . . . . . . . . . . . . . . . . . . . 24
3 Techniques of Confocal Microscopy 27
3.1 Conceptual Basis and Applications . . . . . . . . . . . . . . . . . . . . . . . 27
3.2 Two-Photon Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
3.3 Fluorescent Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
3.3.1 Probes for One-Photon Microscopy . . . . . . . . . . . . . . . . . . . 29
3.3.2 Probes for Two-Photon Microscopy. . . . . . . . . . . . . . . . . . . 30
3.4 Fluorescence Fluctuation Spectroscopy . . . . . . . . . . . . . . . . . . . . . 31
3.4.1 Correlation Function . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
3.4.2 Photon Counting Histogram and Cumulant Analysis . . . . . . . . . 32
IContents
II Experiments 35
4 Experimental Setup 37
4.1 Bulk Spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
4.1.1 Absorption and Fluorescence Spectroscopy . . . . . . . . . . . . . . 37
4.2 Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
4.2.1 Excitation Light Sources . . . . . . . . . . . . . . . . . . . . . . . . . 38
4.2.2 Excitation Arrangement . . . . . . . . . . . . . . . . . . . . . . . . . 39
4.2.3 Microscope Description . . . . . . . . . . . . . . . . . . . . . . . . . 40
4.2.4 Sample Positioning . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
4.2.5 Filter Arrangement Used in Applications . . . . . . . . . . . . . . . 41
4.2.6 Objective Lenses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
4.2.7 Detectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
4.2.8 Computer Hardware and Software . . . . . . . . . . . . . . . . . . . 43
4.2.9 Measurements of Fluorescence Lifetime . . . . . . . . . . . . . . . . 43
4.3 Sample Preparation and Data Analysis . . . . . . . . . . . . . . . . . . . . . 44
4.3.1 Estimation of Bulk Spectral Properties. . . . . . . . . . . . . . . . . 44
4.3.2 Sample Preparation for Low-Temperature Experiments . . . . . . . 44
4.3.3 Sample Preparation for Single-Molecule Experiments . . . . . . . . . 46
4.3.4 Volume Calibration Procedure . . . . . . . . . . . . . . . . . . . . . 47
4.3.5 Calculation of the Excitation Rate . . . . . . . . . . . . . . . . . . . 48
4.3.6 Weighted Averaging of Single-Molecule ACFs . . . . . . . . . . . . . 48
4.3.7 Power Density Correction in Two-Photon Experiments . . . . . . . . 48
4.3.8 Estimation of Bleaching Yields on the Single Molecule Level . . . . . 49
4.3.9 Calculation of the FRET Index and Channel Cross-Talk Correction 49
5 Red Fluorescent Protein eqFP611 51
5.1 Introduction: General Description of eqFP611 . . . . . . . . . . . . . . . . . 51
5.1.1 Spectroscopic Properties . . . . . . . . . . . . . . . . . . . . . . . . . 51
5.1.2 Structural Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
5.2 Experimental Part: Disruption of the Tetramer . . . . . . . . . . . . . . . . 52
5.3 Cryogenic Experiments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
5.4 FCS on Individual Molecules . . . . . . . . . . . . . . . . . . . . . . . . . . 58
5.5 Photostability of Individual eqFP611 Molecules . . . . . . . . . . . . . . . . 61
5.5.1 Molecular Brightness of eqFP611 Tandems . . . . . . . . . . . . . . 61
5.6 Photochromic Properties of eqFP611 . . . . . . . . . . . . . . . . . . . . . . 64
5.6.1 Red-Shifted Variants Show KFP-like Photochromic Properties . . . 64
5.6.2 High-Power Photoconversion . . . . . . . . . . . . . . . . . . . . . . 67
5.7 Spectral Properties of eqFP611 Variants . . . . . . . . . . . . . . . . . . . . 68
5.8 Discussion and Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
5.8.1 eqFP611 versus KFP: Structural Comparison . . . . . . . . . . . . . 70
5.8.2 Precursor Species in eqFP630 Photoisomerization . . . . . . . . . . . 70
5.8.3 Maturation of the eqFP611 Chromophore . . . . . . . . . . . . . . . 70
5.8.4 Protonation State of Mature and Immature Chromophores . . . . . 72
IIContents
5.8.5 Effects at Cryogenic Temperatures . . . . . . . . . . . . . . . . . . . 73
5.8.6 Photodynamics of Fluorescent Proteins . . . . . . . . . . . . . . . . 75
6 Photoconvertible Fluorescent Protein EosFP 77
6.1 Introduction: Overview of Structural Properties . . . . . . . . . . . . . . . . 77
6.1.1 Quaternary Structure . . . . . . . . . . . . . . . . . . . . . . . . . . 77
6.1.2 Molecular Basis of Photoconversion . . . . . . . . . . . . . . . . . . 77
6.2 Experimental Part: Spectroscopic Properties. . . . . . . . . . . . . . . . . . 79
6.2.1 Determination of Conversion Rates . . . . . . . . . . . . . . . . . . . 79
6.2.2 EosFP at Low and High pH . . . . . . . . . . . . . . . . . . . . . . . 83
6.2.3 Fluorescence Lifetime of Green and Red EosFP . . . . . . . . . . . . 85
6.2.4 Excitation and Emission Spectra of wtEosFP at Low Temperatures 85
6.2.5 Spectroscopic Properties of a Non-Convertible EosFP Variant . . . . 87
6.3 Nonlinear Properties of wtEosFP . . . . . . . . . . . . . . . . . . . . . . . . 87
6.3.1 Two-Photon Excitation of the Green Form of wtEosFP . . . . . . . 87
6.3.2 Two-Photon Conversion of wtEosFP . . . . . . . . . . . . . . . . . . 89
6.3.3 Two-Photon Excitabilities of the Red Form of wtEosFP and eqFP611 92
6.4 Experiments on Individual EosFP Molecules . . . . . . . . . . . . . . . . . . 93
6.4.1 Single-Molecule Photoconversion . . . . . . . . . . . . . . . . . . . . 93
6.4.2 Photostability of Individual EosFP Molecules . . . . . . . . . . . . . 94
6.5 Applications of EosFP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
6.5.1 Application of EosFP for Optical Labeling. . . . . . . . . . . . . . . 96
6.5.2 EosFP to Probe Protein-Protein Interactions via FRET . . . . . . . 97
6.5.3 Estimation of EosFP Concentration in HEK293 Cells . . . . . . . . 101
6.6 Other Photoactivable Fluorescent Proteins . . . . . . . . . . . . . . . . . . . 102
6.7 Spectral Properties of EosFP Variants . . . . . . . . . . . . . . . . . . . . . 104
6.8 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
6.8.1 Protonation State of the EosFP Chromophore . . . . . . . . . . . . 104
6.8.2 Vibronic Structure of EosFP Two-Photon Excitation Spectrum . . . 105
Outlook 107
Bibliography 109
Summary 127
Zusammenfassung 131
Achievements 135
Curriculum Vitae 137
Acknowledgments 139
IIIContents
IVList of Figures
0.1 Fluorescent proteins in life science research . . . . . . . . . . . . . . . . . . 2
1.1 Native sources of fluorescent proteins . . . . . . . . . . . . . . . . . . . . . . 8
1.2 The structure of avGFP and its chromophore surrounding . . . . . . . . . . 9
1.3 Formation of GFP and dsRed chromophores . . . . . . . . . . . . . . . . . . 10
1.4 Resonance forms of the GFP chromophore . . . . . . . . . . . . . . . . . . . 11
1.5 Autocatalytic basis of dsRed maturation . . . . . . . . . . . . . . . . . . . . 12
1.6 Spectral properties of avGFP . . . . . . . . . . . . . . . . . . . . . . . . . . 14
1.7 pH-dependence of GFP flickering dynamics . . . . . . . . . . . . . . . . . . 17
2.1 Ground and excited states of a molecule . . . . . . . . . . . . . . . . . . . . 21
2.2 Diagram of a two-photon excitation process . . . . . . . . . . . . . . . . . . 24
3.1 Principle of a confocal system . . . . . . . . . . . . . . . . . . . . . . . . . . 28
3.2 Excitation volumes in the cases of TPE and OPE . . . . . . . . . . . . . . . 29
3.3 Chemical structures of common dyes . . . . . . . . . . . . . . . . . . . . . . 30
3.4 Set of ACFs, presenting light-induced dynamics of eqFP611 . . . . . . . . . 32
4.1 Excitation Arrangement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
4.2 Microscope setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
4.3 Trans