Assessing the functional structure of molecular transporters by EPR spectroscopy [Elektronische Ressource] / Matthias J. N. Junk

Junk

Informations

• Publié par: johannes_gutenberg-universitat_mainz
• Publié le: 01 janvier 2010
• Nombre de lectures: 6
• Langue: English
• Poids de l'ouvrage: 12 Mo

Extrait

Assessing the Functional Structure of Molecular nTsrpaorters
by EPR Spectroscopy



Dissertation
zur Erlangung des Grades

“Doktor der Naturwissenschaften”

im Promotionsfach Chemie
am Fachbereich Chemie, Pharmazie und Geowissenschatefn
der Johannes Gutenberg-Universität
in Mainz



Matthias J. N. Junk
geboren in Bernkastel-Kues



Mainz 2010





























D77 (Dissertation Universität Mainz )
























Gehe nicht, wohin der Weg führen mag,
sondern dorthin, wo kein Weg ist,
und hinterlasse eine Spur.

Jean Paul






Contents
___________________________________________________________________________

Summary xi
1 General Introduction 1
2 Electron Paramagnetic Resonance Theory 11
2.1 Historical Review ……………………………………………………………………………………………….. 11
2.2 EPR Fundamentals …………………………………………………………………………………………… 12
2.2.1 Preface ……………………………………………………………………………………………. 12
2.2.2 Resonance Phenomenon ……………………………………………………………………. 13
2.2.3 Magnetization …………………………………………………………………………………. 14
2.2.4 Bloch Equations ……………………………………………………………………………………. 15
2.2.5 Continuous Microwave Irradiation ………………………………………………….. 17
2.3 Types of Interactions and Spin Hamiltonian …………………………………………………..20
2.3.1 Electron Zeeman Interaction ………………………………………………………… 21
2.3.2 Nuclear Zeeman Interaction …………………………………………………………. 22
2.3.3 Hyperfine Interaction ……………………………………………………………………………. 22
2.3.4 Nuclear Quadrupole Interaction …………………………………………………………... 24
2.3.5 Nuclear Spin–Spin Interaction ………………………………………………………. 24
2.3.6 Zero-Field Splitting ……………………………………………………………………….. 24
2.3.7 Weak Coupling between Electron Spins ……………………………………….. 25
2.4 Anisotropy in EPR Spectra …………………………………………………………………………….. 27
2.4.1 Anisotropy …………………………………………………………………………………………. 27
2.4.2 Combined Anisotropies in Real Spectra …………………………………………… 29
2.5 Dynamic Exchange ………………………………………………………………………………………….. 31
2.6 Nitroxides as Spin Probes ……………………………………………………………………………… 34
2.6.1 Spin Probe vs. Spin Label ……………………………………………………………. 35
2.6.2 Quantum Mechanical Description ………………………………………………………… 36
2.6.3 Nitroxide Dynamics ………………………………………………………………………………. 38
2.6.4 Environmental Influences ……………………………………………………………….. 40
2.7 CW Spectral Analysis via Simulations ………………………………………………………………. 42
2.8 Time Evolution of Spin Ensembles ……………………………………………………………… 43 vi Contents

2.8.1 The Density Matrix ………………………………………………………………………….. 43
2.8.2 Product Operator Formalism …………………………………………………………… 45
2.8.3 Application to EPR ………………………………………………………………………………… 46
2.8.4 The Vector Model …………………………………………………………………………………. 47
2.8.5 The ( 1/2, 1/2) Model System …………………………………………………… 47
2.9 Pulse EPR Methods Based on the Primary Ec…ho… …………………………………………. 49
2.9.1 ESE Detected Spectra ……………………………………………………………………. 51
2.9.2 2-Pulse Electron Spin Echo Envelope Modiounla (tESEEM) …………………… 51
2.10 Pulse EPR Methods Based on the Stimulated oE …ch………………………………………….. 53
2.10.1 3-Pulse ESEEM ……………………………………………………………………………… 54
2.10.2 Hyperfine Sublevel Correlation (HYSCORE) pSectroscopy ……………………… 55
2.10.3 Blind Spots ……………………………………………………………………………………. 57
2.10.4 Phase Cycling …………………………………………………………………………………… 58
2.11 Double Electron–Electron Resonance ………………………………………………………………… 59
2.12 Bibliography ……………………………………………………………………………………………………. 63
2.13 References ……………………………………………………………………………………………………. 63
3 The Functional Structure of Human Serum Albumin 7 6
3.1 Introduction …………………………………………………………………………………………………. 68
3.2 The Distribution of Fatty Acids in Human Sem ru Albumin in Solution ………………… 71
3.2.1 Results …………………………………………………………………………………………….. 71
3.2.2 Discussion ……………………………………………………………………………………….. 74
3.3 Multispin Contributions to DEER Spectra …………………………………………………… 78
3.3.1 Spin Counting ……………………………………………………………………………….. 78
3.3.2 Quantification of Multispin Artifacts ……………………………………………………. 82
3.4 Orientation Selectivity in DEER: Beyond nDciestsa …………………………………………….. 86
3.5 Conclusions …………………………………………………………………………………………………….. 95
3.6 Materials and Methods ……………………………………………………………………………… 96
3.7 References ……………………………………………………………………………………………………. 99
4 Copper Complexes of Star-Shaped Cholic Acoimd eOrsl ig
with 1,2,3-Triazole Moieties 103
4.1 Introduction ………………………………………………………………………………………………….. 104
4.2 Triazole-Substituted Oligomer T3t as Metal SIeonsor ……………………………………. 108
4.2.1 Fluorescence Quenching Enhancement by T3……t ………………………………. 108
4.2.2 T3t – A Tridentate Ligand for Copper ………………………………………………….. 110
4.2.3 Deriving a Molecular Picture from the eEsPuRlt sR …………………………….. 115
4.2.4 Comparative Fluorescence Quenching Studieosf T3t and T0 ……………… 116
4.2.5 Summary ………………………………………………………………………………………….…. 117 Contents vii


4.3 Influence of the Molecular Structure on thtea lM Ioen Complexation ……………… 118
4.3.1 Fluorescence Quenching of T3b ………………………………………………………….. 118
4.3.2 Copper Coordination in T3b ………………………………………………………………… 119
4.3.3 Influence of a Fourth Cholic Acid Arm …………………………………………. 124
4.3.4 Observation of a Fourth Species in a 2x:t1u rMe iof CuCl and Q8tb …… 126 2
4.3.5 Summary ……………………………………………………………………………………….….… 127
4.4 Self-Assembly of Monomeric Cholic Acid Derivaivtes ………………………………………. 128
2+ 4.4.1 Cu Coordination by S2 ……………………………………………………………………… 128
2+
4.4.2 Cu Mediated Assembly of Single Cholic Acid Chains …………………………. 131
4.5 Conclusions …………………………………………………………………………………………………… 135
4.6 Materials and Methods ………………………………………………………………………………. 136
4.7 References ……………………………………………………………………………………………………. 138
5 Nano-Inhomogeneities in Structure and React ivity
of Thermoresponsive Hydrogels 141
5.1 Introduction ………………………………………………………………………………………………….. 142
5.2 Results …………………………………………………………………………………………………………… 144
5.2.1 The Temperature Induced Hydrogel Collap se
as Seen by Probe Molecules …………………………………………………………………… 144
5.2.2 Chemical Decomposition of Spin Probes in
Hydrophilic Regions of the Hydrogel …………………………………………………… 149
5.3 Discussion …………………………………………………………………………………………………… 152
5.3.1 The Hydrogel Collapse on a Molecular L…ev…e…l ………………………………….. 152
5.3.2 Hydrogel Inhomogeneities on the Nanosceaaled Lto
Nanoreactors and Nanoshelters ……………………………………………………….... 155
5.3.3 Nanoreactors with Localized Acid Groups ……………………………………… 156
5.4 Conclusions …………………………………………………………………………………………………… 158
5.5 Materials and Methods ………………………………………………………………………………. 159
5.6 References ………………………………………………………………………………………………………. 160
6 Thermoresponsive Spin-Labeled Hydrogels as Sebplea raDNP Polarizing Agents 163
6.1 Introduction ……………………………………………………………………………………………….. 164
6.2 Theory of Overhauser DNP …………………………………………………………………………. 166
6.3 Results and Discussion ………………………………………………………………………………168
6.3.1 Strategy for the Preparation of
Thermoresponsive Spin-Labeled Hydrogels …………………………………………. 168
6.3.2 CW EPR Characterization of the Spin-Lab Heylderdogels ……………………….. 170
1 6.3.3 Characteristic DNP Factors aH nRde laxation Times ……………………………… 171
6.3.4 Temperature-Dependent DNP Performancep ino-f LSabeled Hydrogels … 173 viii Contents

6.4 Conclusions …………………………………………………………………………………………………… 175
6.5 Outlook …………………………………………………………………………………………………………. 176
6.6 Materials and Methods ……………………………………………………………………………………. 177
6.7 References ……………………………………………………………………………………………………. 179
7 Local Nanoscopic Heterogeneities in Thermoresipvoen sDendronized Polymers 183
7.1 Introduction ………………………………………………………………………………………………….. 184
7.2 Local Heterogeneities above the Critical Tempreature …………………………………….. 187
7.3 Pre-Collapse of the Dendronized Polymers be ltohwe Critical Temperature ……. 194
7.4 Formation and Influence of a Dense Periph ePraolymeric Layer ……………………… 196
7.4.1 Results ……………………………………………………………………………………………… 196
7.4.2 Discussion ………………………………………………………………………………………… 200
7.5 Conclusions …………………………………………………………………………………………………… 206
7.6 Materials and Methods ……………………………………………………………………………………. 208
7.7 References ……………………………………………………………………………………………………. 209
8 Conclusion 213
Appendix 217
A The Functional Structure of Human Serum Albumin ………………………………………… 217
A.1 Reduction of Spin-Labeled Fatty Acids to EIPnRa-ctive Hydroxylamines … 217
A.2 CW EPR Studies with Paramagnetic DSA withouStp in Dilution ……………… 220
A.3 CW EPR Studies of Fatty Acid Free HSA ……………………………………………….. 221
A.4 No Dimers in Solution Observed by DEER ………………………………………. 223
A.5 Background Correction ………………………………………………………………………….. 224
A.6 Distances Retrieved f