목차

I. Introduction 1
1. Nuclear Magnetic Resonance (NMR) Spectroscopy 1
1) Basic of NMR 1
2) Chemical shift 2
2. Biomolecular NMR Spectroscopy 3
1) Nucleic Acid 3
2) Protein 6
3) Protein Dynamics 13
3. History of Z-DNA 20
4. Z-DNA Binding Proteins 21
II. Solution structure of the Z-DNA binding domain of PKR-like protein kinase from Carassius auratus and quantitative analyses of the intermediate complex during B-Z transition 26
1. Introduction 26
2. Purpose of the Experiment 29
3. Materials and Method 32
1) Sample preparation 32
2) NMR Experiments 34
3) Solution Structure Calculation 36
4) Nitrogen Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion 36
5) Binding Models 37
6) Hydrogen Exchange rate 41
3. Results 43
1) Solution structure of free caZαPKZ 43
2) Titration of caZαPKZ into dT(CG)₃under various [NaCl] 45
3) Chemical shift changes in caZαPKZ upon binding to dT(CG)3 47
4) Chemical shift differences in caZαPKZ bound to B-DNA and Z-DNA 53
5) 15N Carr-Purcell-M eiboom-Gill (CPMG) relaxation dispersion experiment on caZαPKZ bound to dT(CG)3 57
6) H-bonding interaction of K56 with Z-DNA phosphate 60
4. Conclusion 63
Ⅲ. NMR elucidation of reduced B-Z transition activity of PKZ protein kinase at high NaCl concentration 67
1. Purpose of the Experiment 67
2. Materials and Method 68
1) Sample preparation 68
2) NMR Experiments 68
3) Global fitting 69
3. Results 70
1) Relative Z-DNA population based on the analysis of imino proton spectra of dT(CG)₃upon binding to caZαPKZ at 250 mM NaCl 70
2) Chemical shift perturbation of caZαPKZ upon binding to dT(CG)₃at 250 mM NaCl 72
3) The dissociation constants for BP and ZP₂at 250 mM NaCl 75
4) Chemical shift differences in caZaPKZ upon binding to B-DNA and Z-DNA 75
5) The salt dependence of B-Z transition activity of caZαPKZ 78
4. Conclusion 79
Ⅳ. NMR dynamics study of the Z-DNA binding domain of human ADAR1 bound to various DNA duplexes 80
1.Introduction 80
2. Purpose of the Experiment 81
3. Materials and Method 82
1) Sample preparation 82
2) NMR Experiments 84
4. Results 85
1) Chemical shift perturbation of hZαADAR1 upon binding to CG6 85
2) Chemical shift changes of hZαADAR1 upon binding to non CG-repeat DNA 87
3) Relaxation rate constants of the amide protons of hZαADAR1 in the free and in a complex with DNA 90
4) Implication for the BZ transition mechanism 93
4. Discussion 96
Ⅴ. Zα domain of ADAR1 prefers to bind to Z-RNA rather than Z-DNA 97
1. Introduction 97
2. Purpose of the Experiment 100
3. Materials and Method 104
1) Sample preparation 104
2) NMR Experiments 104
3) Theoretical NMR lineshapes for twosite exchange 105
4) Nitrogen Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion 109
5) 15NZ-Exchange Experiment 110
4. Results and Discussions 111
1) Titration of hZαADAR1 into d(CG)₃and r(CG)3 111
2) Comparison of binding modes of hZαADAR1 toZ-DNA and Z-RNA 115
3) Chemical shift changes in hZαADAR1 upon binding Z-DNA 119
4) Chemical shift changes in hZαADAR1 upon binding to Z-RNA 123
5) 15N CPMG Relaxation Dispersion Experiment of ZαADAR1 Bound to d(CG)3 124
6) Unusual Exchange Behavior of E171, N173, and Y 177 Amide Protons 125
7) 15N CPMG Relaxation Dispersion Experiment of ZαADAR1 Bound to r(CG)3 127
8) Conformational Differences of hZαADAR1 Between Free and Bound States and Between A/B-form and Z-form Bound States 132
9) 5Nz-exchange Experiment on hZαADAR1 Bound to r(CG)3 132
Ⅵ. Reference 134