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title page
Contents
Introduction 11
PART I. The structure and proteolytic mechanism of ClpX and ClpP 13
1.0. ABSTRACT 13
SUBPART I. Crystal structure of ClpX molecular chaperone from Helicobacter pylori 15
1.1.1. Introduction 15
1.1.2. Experimental procedures 18
1.1.2.1. Protein Preparation and Crystallization 18
1.1.2.2. The Size Measurement of Hp ClpX-ASD 18
1.1.2.3. Data Collection, Structure Determination, and Refinement 19
1.1.2.4. Modeling of Hexameric ClpX-ASD 20
1.1.3. Results and discussion 21
1.1.3.1. Size Determination of Hp ClpX-ASD in Solution 21
1.1.3.2. Overall Structure of Hp ClpX-ASD 23
1.1.3.3. Structural Comparison with Homologous Proteins 25
1.1.3.4. Hexameric Assembly of Hp ClpX-ASD 30
1.1.3.5. Nucleotide Binding Site 32
1.1.4. Conclusion 37
1.1.5. REFERENCES 38
SUBPART II. Activation mechanism of ClpP protease 43
1.2.1. Introduction 43
1.2.2. Materials and methods 46
1.2.2.1. HpClpP expression and preparation 46
1.2.2.2. Crystallization and data collection 46
1.2.2.3. Structure determination and refinement 47
1.2.2.4. Isothermal titration calorimetry(내용없음) 48
1.2.3. Results 49
1.2.3.1. Structure determination of HpClpP 49
1.2.3.2. Overall structure of HpClpP 49
1.2.3.3. Crystal structure of HpClpP in complex with peptides 53
1.2.3.4. ClpP recognizes substrate via backbone interaction 54
1.2.3.5. Substrate binding pockets of HpClpP 58
1.2.3.6. Substrate binding hastens rearrangement of active sites 61
1.2.4. Discussion 65
1.2.4.1. Non-specific hydrophobic interaction between ClpX and ClpP 65
1.2.4.2. The sequential digestion and release of a substrate by ClpP 67
1.2.4.3. The structural implication of the communication between ClpX and ClpP 71
1.2.5. REFERENCES 72
PART II. The mechanism for the temperature induced functional conversion of bacterial HtrA 77
2.1. ABSTRACT 77
2.2. Introduction 78
2.3. Experimental procedures 81
2.3.1. Plasmid construction and site directed mutagenesis for EPR spectroscopy 81
2.3.2. Protein expression, purification, and spin labeling 81
2.3.3. EPR data collection 82
2.3.4. Assay of peptidase activity 82
2.3.5. Circular dichroism 83
2.3.6. The size measurement of HtrA mutants 83
2.3.7. Acrylamide quenching 83
2.4. Results 85
2.4.1. The formation of quality control chamber by PDZ2 85
2.4.2. Lineshape analysis of room temperature EPR spectra 87
2.4.3. Distance measurement 91
2.4.4. Temperature-dependent flexibility change depending on position 92
2.4.5. Local environmental change paralleling with function conversion 98
2.4.6. Structure of HL at high temperature(이미지참조) 101
2.5. Discussion 104
2.6. REFERENCES 107
PART III. The structural and functional study of E.coli RseB, regulator of oE signaling.(이미지참조) 110
3.1. Introduction 110
3.2. Materials and methods 113
3.2.1. Cloning, expression and protein purification of RseB 113
3.2.2. Crystallization 113
3.2.3. Structure determination and refinement 114
3.2.4. Dimerization by cysteine mutation of RseB 115
3.2.5. His-tag pull-down assay 115
3.2.6. Size exclusion chromatography 116
3.2.7. Circular dichroism 116
3.3. Results and discussion 118
3.3.1. Structure of RseB monomer 118
3.3.2. RseB dimer of two conformations 123
3.3.3. RseB interacts with binding motif of RseA strongly via charge interaction 127
3.3.4. RseB binding motif of RseA has helical conformation 128
3.3.5. The Closed RseB does not interact with RseA 133
3.4. REFERENCES 134
[요약] 139
Table 1a-I. Data collection and refinement statistics of Hp ClpX-ASD 24
Table 1b-I. Data collection and refinement statistics 48
Table 1b-II. The distances of catalytic triads of HpClpP and HpClpP-NVLGFTQ complex 63
Table 1b-III. The distances of catalytic triads of HpClpP and HpClpP-AAAA complex 64
Table 2-I. Oligomerization of T.mar HtrA mutants 86
Table 2-II. Ksv values of acrylamide quenching(이미지참조) 97
Table 3-I. Data collection and refinement statistics of E.coli RseB 117
PART I. 8
Fig 1a-1. Size determination of Hp ClpX-ASD. 22
Fig 1a-2. The structures of Hp ClpX-ASD, E.coli HslU and E. coli ClpA D2. 27
Fig 1a-3. The hexamer model of Hp ClpX-ASD and E.coli ClpP heptamer. 34
Fig 1a-4. Stereo view of nucleotide binding region of Hp ClpX. 36
Fig 1b-1. The structure of H.pylori ClpP. 51
Fig 1b-2. Diverse electron densities of hepta-peptide (NVLGFTQ) bound to HpClpP 55
Fig 1b-3. The substrate recognition of HpClpP 56
Fig 1b-4. The substrate binding pocket of HpClpP. 59
Fig 1b-5. Activation of HpClpP by the binding of substrates. 60
Fig 1b-6. Additional electron density of binding cleft in the structure of DFP-modified HpClpP/peptide NVLGFTQ complex. 66
Fig 1b-7. The sequential digestion of substrate by ClpP. 69
Fig 1b-8. The model of ClpXP communication. 70
PART II. 8
Fig 2-1. A. Proteolytic activities of native and reduced α-lactalbumin. The molar ratio of T.mar HtrA to native α-lactalbumin (or reduced form) was adjusted to 1:150. B. Mutation sites for EPR spectroscopy in full length HtrA.... 88
Fig 2-2. Distance measurement of double labeled mutants. 90
Fig 2-3. Not overall structural perturbation but position-dependent temperature sensitivity of flexibility responsible for the HL opening.(이미지참조) 94
Fig 2-4. Fluorescence quenching depending on position at various temperatures. 95
Fig 2-5. Local environmental change near active site paralleling with functional conversion. 99
Fig 2-6/6. The structure of the removed HL and a proposed model for HtrA function.(이미지참조) 102
PART III. 9
Fig 3-1. Structure of the E.coli RseB monomer. 120
Fig 3-2. Interaction between large and small domain in closed (A) and open (B) RseB. 122
Fig 3-3. Butterfly-like structure of RseB dimer. 125
Fig 3-4. The mapping of RseA domain for RseB binding. 129
Fig 3-5. RseA·B binding via charge interaction. 130
Fig 3-6. Circular dichroism spectra of RseA169-186(이미지참조). 132
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