Spectroscopic studies of ligand migration in myoglobin [Elektronische Ressource] / Karin Nienhaus

Knienhau

Sujets

• Biologie

Informations

• Publié par: universitat_ulm
• Publié le: 01 janvier 2003
• Nombre de lectures: 22
• Langue: English
• Poids de l'ouvrage: 41 Mo

Extrait

Spectroscopic Studies
of
Ligand Migration
in
Myoglobin











Dissertation zur Erlangung des Doktorgrades Dr. rer. nat.
der Fakultät für Naturwissenschaften
der Universität Ulm





vorgelegt von Karin Nienhaus
Rhede
2002



























Amtierender Dekan: Prof. Dr. Wolfgang Witschel

Erstgutachter: Prof. Dr. Peter Bäuerle

Zweitgutachter: Prof. Dr. Hartmut Seliger

Tag der Promotion: 13.02.2003

Contents:


1. Introduction ............................................................................................................. 1
1.1 Protein Structure, Function, Dynamics ............................................................. 1
1.2 Myoglobin, the Hydrogen Atom of Biology..................... 3
Brief Historical Overview ................................................................................. 3
1.2.1 Structure and Function.......... 3
1.2.2 Ligand Binding and Protein Dynamics................. 5
1.2.3 Infrared Spectroscopy............................................................................ 13
1.2.4 Outline of this work............... 17


2. Materials and Methods ............................................................................................ 19
2.1 Protein Expression and Purification.................................. 19
2.1.1 Transformation and Multiplication........................ 19
2.1.2 Lysis and Purification............................................................................ 19
2.2 Mutagenesis....................................... 20
2.3 Sample Preparation............................................................ 22
2.4 Absorption Spectroscopy................................................... 23
2.4.1 Introduction........................... 23
2.4.2 UV/Visible Spectroscopy ...................................................................... 25
2.4.2.1 Survey Spectra in the Visible.......................... 25
2.4.2.1 Flash Photolysis............... 25
2.5 Cryospectroscopy in the Infrared...... 25
2.6 Fourier Transform Infrared (FTIR) Techniques................................................ 26
2.6.1 FTIR Absorbance Difference Spectra................... 27
2.6.2 Isothermal Kinetic Measurements......................... 29
2.6.3 Temperature Derivative Spectroscopy (TDS) ...................................... 29
2.6.3.1 Theoretical Background .................................. 33
2.6.3.2 Kinetic Hole Burning in the TDS Map............ 35
2.6.3.2.1 Temperature-dependent Transmission Spectra and TDS Maps ...... 36
2.6.4 Illumination Protocols ........................................................................... 39


3. Results and Interpretation....................................................................................... 41
3.1 WT and Related Mutants................... 41
3.1.1 Probing Active Site and Initial Docking Site ........................................ 41
3.1.1.1 FTIR Absorbance Spectra after Short Illumination......................... 41
3.1.1.2 TDS Contour Maps after Short Illumination... 46
i 3.1.1.3 Discussion: Bound State A and Initial Docking Site B ................... 52
3.1.2 Probing Intermediates within the Xe non cavities .................................. 55
3.1.2.1 FTIR Absorbance Spectra after Slow Cooling from 160 to 3 ........ 55
3.1.2.2 TDS after Slow Cooling from 160 to 3 K....... 59
3.1.2.3 Interpretation and Discussion:
TDS after Slow Cooling from 160 to 3 K ....................................... 64
3.1.3 Probing Intermediates above the Dynamic Transition Temperature .... 72
3.1.3.1 FTIR Absorbance Spectra at 160 K................. 72
3.1.3.2 TDS after Illumination at 185 K and
Slow Cooling from 185 to 160 K .................................................... 74
3.1.3.3 Interpretation and Discussion: Intermediate States above
the Dynamic Transition Temperature.............. 76

3.2 Mutant L29W and Related Double Mutants ..................................................... 78
3.2.1 Probing Taxonomic Substates A and A.............. 79 I II
3.2.1.1 FTIR Absorbance Difference Spectra at 3 K .................................. 79
3.2.1.2 Interpretation and Discussion: Interconversion between
Taxonomic Substates A and A ...................................................... 83 I II
3.2.2 Probing the Initial Docking Site B........................ 86
3.2.2.1 FTIR Absorbance Difference Spectra after Short Illumination
for 1 s at 3 K .................................................... 86
3.2.2.2 TDS after Short Illumination at 3 K ............................................... 88
3.2.2.3 Interpretation and Discussion: .................. 91
3.2.3 Remote Intermediates: Site C’............................................................... 93
3.2.3.1 Isothermal Illumination at 3 K........................ 93
3.2.3.2 TDS Experiment after Isothermal Illumination at 3 K .................... 94
3.2.3.3 Isothermal Illumination at 80 K ...................................................... 95
3.2.3.4 Interpretation and Discussion:
Structural Interpretation of Intermediate Site C’ ............................. 96
3.2.4 Detailed Investigations of Photoproduct State C’ reveal an Additional
Photoproduct State C’’………………………………………………. 101
3.2.4.1 Isothermal Illumination at 120 K ....................................................101
3.2.4.2 FTIR Absorbance Difference Spectra after Slow Cooling
from 160 to 3 K ...............................................102
3.2.4.3 TDS after Slow Cooling from 160 to 3 K .......................................104
3.2.4.4 Isothermal Illumination at 160 K....................108
3.2.4.5 TDS after Isothermal Illumination at 160 K....109
3.2.4.6 TDS after Slow Cooling from 160 to 100 K...110
3.2.4.7 Interpretation and Discussion: Structural Interpretation of
Intermediate Site C’’ .......................................................................111
3.2.5 Intermediate D, the Photoproduct in a Fluctuating Protein...................114
3.2.5.1 TDS under Illumination...114
3.2.5.2 Isothermal Kinetics at 180 K ...........................................................115
3.2.5.3 FTIR Absorbance Difference Spectra after Illumination
at 185 K and Slow Cooling under Light to 160 K..........................117
3.2.5.4 TDS after Illumination at 185 K and Slow Cooling to 160 K .........119
ii 3.2.5.5 Interpretation and Discussion: Photoproducts in a Fluctuating
Protein ..............................................................................................122


4. The Role of Internal Cavities in Physiological Ligand Binding...........................126
4.1 Migration and Recombination in WT MbCO ...................................................126
4.1.1 The Primary Docking Site B.................................126
4.1.2 Ligand Migration across a One -dimensional Energy Landscape ..........127
4.1.3 Physiological Ligand Binding in WT MbCO........133
4.2 Ligand Migration and Recombination in L29W MbCO ...................................141
4.2.1 Ligand Dynamics in a Frozen Protein ...................................................141
4.2.2 Physiological Ligand Binding in L29W MbCO....144
4.3 Conclusion .........................................................................................................147

Appendix A: Intrinsic Absorbance Changes of Photoproduct Bands. i

Appendix B: CO Stretch Frequencies ................................................................................ viii

Appendix C: List of Figures................................................................................................ ix

Bibliography ....................................................... xiv

Summary.............................................................................................. xix

Zusammenfassung............................................................................................................... xxii

List of Publications............. xxvi

Acknowledgements .............................................................................................................xxviii

Curriculum Vitae.................xxix


iii 1. Introduction

1.1 Protein Structure, Dynamics and Function
Proteins are the working machines of all living systems. Antibodies, for example, protect
from dis