Title Page
Contents
ABBREVIATIONS 7
ABSTRACT 15
PART I. OVERVIEW 17
1. Intrinsically Unfolded Proteins 17
1.1. The origin of intrinsically unfolded proteins 17
1.2. Scientific discovery of the IUP 17
1.3. What is the IUP term? 21
1.4. Functional feature of IUPs 23
1.5. Interrelation between the IUPs and various diseases. 24
1.6. The IUP in protein networks 25
1.7. The binding mechanisms 26
2. NMR spectroscopy 29
2.1. A short history of NMR 29
2.2. Theory of NMR 31
2.3. Boltzmann statistics 33
2.4. General experimental scheme 34
2.5. The HSQC pulse sequence 36
2.6. The examples of Pulse sequence for 3D NMR 38
2.7. NMR in IUPs 39
PART II. HSV VP16 TAD 41
II-1. INTRODUCTION 41
II-2. MATERIALS AND METHODS 44
1. Materials 44
2. Secondary structure prediction 44
3. PCR amplification and subcloning of VP16 TAD (412-490) 45
4. Expression and purification of VP16 TAD (412-490) 46
5. Peptide synthesis and purification 48
6. Circular dichroism (CD) spectropolarimeter 50
7. Surface plasmon resonance (SPR) 50
8. Nuclear magnetic resonance (NMR) spectroscopy 51
II-3. RESULTS AND DISCUSSION 53
1. Secondary structure prediction results of VP16 TAD 53
2. PCR amplification and subcloning of VP16 TAD 56
3. Expression and purification of VP16 TAD in E. coli 57
4. CD spectra of the VP16 TAD 62
5. SPR analysis 63
6. NMR spectra of VP16 TAD 64
II-4. CONCLUSIONS 70
PART III. HBV preS1 71
III-1. INTRODUCTION 71
III-2. MATERIALS AND METHODS 74
1. Materials 74
2. Secondary structure prediction 74
3. Expression and purification of HBV preS1 (1-119) from E. coli 74
4. Peptide preparation of preS1 (21-47) 75
5. NMR spectroscopy 75
III-3. RESULTS AND DISCUSSION 76
1. Secondary structure prediction results of HBV preS1 76
2. Expression and purification of preS1 (1-119) (adr subtype) 78
3. Peptide synthesis and purification of preS1 (21-47) 81
4. NMR spectra of preS1 82
III-4. CONCLUSIONS 91
PART IV. HIF-1α ODD 92
IV-1. INTRODUCTION 92
IV-2. MATERIALS AND METHODS 94
1. Materials 94
2. Secondary structure prediction 94
3. Expression and purification of HIF-1α ODD from E. coli 94
3.1. Expression and purification of HIF-1α ODD (403-603) 94
3.2. Expression and purification of HIF1α ODD_N (404-477) 95
4. NMR spectroscopy 96
IV-3. RESULTS AND DISCUSSION 97
1. Secondary structure prediction results of HIF-1α ODD (403-603) 97
2. Expression and purification of HIF-1α ODD (403-603) 100
3. Expression and purification of HIF-1α ODD_N (404-477) 104
4. NMR spectra of HIF1α ODD 110
IV-4. CONCLUSIONS 114
PART V. DISCUSSION 115
PART VI. PERSPECTIVE 116
PART VII. REFERENCES 117
SUMMARY (IN KOREAN) 146
Table 1. Predictors of intrinsically unfolded proteins. 19
Table 2. Values for some common nuclei in NMR spectroscopy 32
Table 3. Classification of human herpes viruses. 41
Fig. 1. Growth in IUP literature 18
Fig. 2. Structural type of IUPs. 21
Fig. 3. Existing significance of the IUP in the protein network. 25
Fig. 4. Basic pulse sequence for 1D, 2D, and 3D acquision. 34
Fig. 5. General experimental scheme for 2D NMR 35
Fig. 6. Pulse sequence for the HSQC experiment. 37
Fig. 7. Pulse sequence for (A) the 3D TOCSY-HSQC and (B) the 3D NOESY-HSQC experiments. 38
Fig. 8. Functional domains of HSV VP16. 42
Fig. 9. Secondary structure prediction for VP16 TAD. 54
Fig. 10. Secondary structure prediction of VP16 TAD (412-490) using IUPred and Agadir. 55
Fig. 11. Construction of bacterial expression vectors for the production of VP16 TAD (412-490). 56
Fig. 12. Overexpression of GST-tagged VP16 TAD (412-490). 57
Fig. 13. Purification of VP16 TAD (412-490) by a Q-Sepharose column. 58
Fig. 14. Purification of VP16 TAD (412-490) by a source 15Q column. 59
Fig. 15. Gel filtration chromatography and SDS-PAGE of VP16 TAD (412 -490). 60
Fig. 16. MALDI-TOF mass spectrum of the purified VP16 TAD (412-490). 61
Fig. 17. Circular dichroism spectra of 10 μM VP16 TAD (412-490). 62
Fig. 18. Biosensorgrams of binding analysis of VP16 TAD peptide with hTAFll31.(이미지참조) 63
Fig. 19. 2D 15N-¹H HSQC spectrum of the full-length VP16 TAD (412-490).(이미지참조) 65
Fig. 20. Summary of interproton NOEs' ³JHNHd' and chemical shift index for the VP16 TAD (412-490).(이미지참조) 66
Fig. 21. Backbone dynamics of VP16 TAD. 67
Fig. 22. 2D 15N-¹H HSQC spectra and chemical shift perturbation of the VP16 TAD titrated with hTAFll31 (1-140).(이미지참조) 68
Fig. 23. Location of PreSMos and target binding sites in VP16 TAD. 69
Fig. 24. Geographic pattern of HBV infection display. 72
Fig. 25. The domain of HBV surface protein. 73
Fig. 26. Secondary structure prediction for HBV preS1 using GOR4 and Jpred3. 76
Fig. 27. Secondary structure prediction of HBV preS1 using IUPred and Agadir. 77
Fig. 28. Overexpression of GST-tagged preS1 (1-119) and GST-tagged preS1 (1-119) purified by glutathione Sepharose beads. 79
Fig. 29. Thrombin digestion of preS1 (1-119). 79
Fig. 30. Gel filtration chromatography of preS1 (1-119). 80
Fig. 31. The spectrum of preS1 (21-47) on a C18 column using RP-HPLC.(이미지참조) 81
Fig. 32. 2D 15N-¹H HSQC spectrum of the preS1 (1-119).(이미지참조) 83
Fig. 33. Summary of ³JHNHa' interproton NOEs' and chemical shift index (CSI) of Ha protons for preS1 (1-119).(이미지참조) 84
Fig. 34. Ha and Ca chemical-shift deviation and temperature coefficient of preS1 (1-119).(이미지참조) 85
Fig. 35. Plots of backbone 15N-¹H heteronuclear NOEs and 15N relaxation times against the residue number of preS1 (1-119).(이미지참조) 86
Fig. 36. Location of pre-structured regions and functional domains in preS1. 87
Fig. 37. Summary of interproton NOEs, ³JHNHa, and chemical shift index (CSI) of Ha protons for preS1 (21-47) peptide.(이미지참조) 89
Fig. 38. HBsAg secretion of HepaRG cells infected by HBV in the presence of preS1 derived peptides. 90
Fig. 39. Domains and potential functions of HIF-1α. 92
Fig. 40. Secondary structure prediction for HIF-1α ODD (403-603) using GOR4 and Jpred3. 98
Fig. 41. Secondary structure prediction of HIF-1α ODD (403-603) using IUPred and Agadir. 99
Fig. 42. Overexpression of HIF-1α ODD (403-603). 100
Fig. 43. Purification of HIF-1α ODD (403-603) by a Q-Sepharose column. 101
Fig. 44. Purification of HIF-1α ODD (403-603) by a DEAE-Sepharose column. 102
Fig. 45. Gel filtration chromatography of HIF-1α ODD (403-603). 103
Fig. 46. Overexpression of HIF-1α ODD_N (404-477). 105
Fig. 47. Purification of HIF-1α ODD_N (404-477) by a Q-Sepharose column. 106
Fig. 48. Gel filtration chromatography of HIF-1α ODD_N (404-477). 107
Fig. 49. Purification of HIF-1α ODD_N (404-477) by a DEAE-Sepharose column. 108
Fig. 50. MALDI-TOF mass spectrum of the purified HIF-1α ODD_N (404-477). 109
Fig. 51. 2D 15N-¹H HSQC spectrum of HIF-1α ODD(403-603).(이미지참조) 111
Fig. 52. 2D 15N-¹H HSQC spectrum of HIF-1α ODD_N (404-477).(이미지참조) 112
Fig. 53. The chemical shift deviations in ¹³Cα, ¹³CO, and Hα for HIF1α ODD_N (404-477).(이미지참조) 113