| 摘要 | 第1-5页 |
| ABSTRACT | 第5-7页 |
| Content | 第7-10页 |
| Chapter 1 Introductions | 第10-24页 |
| ·Introduction of ISC proteins in Acidithiobacillus ferrooxidans | 第10-15页 |
| ·Introduction of Acidithiobacillus ferrooxidans | 第10页 |
| ·Introduction of Iron-sulfur cluster biosynthesis systeim | 第10-11页 |
| ·The cysteine desulfurase IscS | 第11-12页 |
| ·The scaffold protein of IscA and IscU | 第12-13页 |
| ·The repressor protein of IscR,ferredoxin and chaperone proteins HscB/HscA | 第13-15页 |
| ·Structure,function,and formation of iron-sulfur clusters | 第15-22页 |
| ·Structures of iron-sulfur clusters | 第15-18页 |
| ·Functions of of iron-sulfur clusters | 第18-20页 |
| ·Formation of iron-sulfur clusters | 第20-22页 |
| ·The objective and main content of the present study | 第22-24页 |
| ·The objective of the present study | 第22-23页 |
| ·The main content of the present study | 第23-24页 |
| Chapter 2 Materials and Methods | 第24-36页 |
| ·Materials | 第24页 |
| ·Methods | 第24-30页 |
| ·Design of Primer of IscS from A.ferrooxidans for pLM1 Vector | 第24-25页 |
| ·PCR amplification | 第25页 |
| ·Purification of PCR Product | 第25页 |
| ·Double Digestion of PCR Product and pLM1 Vector | 第25-26页 |
| ·Purification of Digested PCR Product and pLM1 Vector | 第26-27页 |
| ·Ligation Reaction | 第27页 |
| ·Transformation and Identification of Positive Colony | 第27-28页 |
| ·Expressive transformation of IscS protein | 第28-29页 |
| ·Expression of soluble recombinant protein IscS in E.coli | 第29页 |
| ·Purification of A.ferrooxidans ATCC 23270 IscS | 第29-30页 |
| ·Site-Directed Mutagenesis | 第30-32页 |
| ·Design of mutant Primer of A.ferrooxidans IscS | 第30页 |
| ·PCR Reaction | 第30-31页 |
| ·Transformation and Identification of Positive Colony | 第31-32页 |
| ·Expression and purification of IscS Mutant Proteins in E.coli | 第32页 |
| ·Analytic method | 第32-33页 |
| ·SDS-polyacrylamide gel electrophoresis(SDS-PAGE) | 第32页 |
| ·UV-visible spectra scanning | 第32页 |
| ·Molecular structure modeling of IscS from A.ferrooxidans~T | 第32-33页 |
| ·EPR spectra | 第33页 |
| ·Enzyme kinetics | 第33-35页 |
| ·Assay of A.ferrooxidans IscS wild type and the mutant proteins activity | 第33-34页 |
| ·Effect of pH and temperature | 第34页 |
| ·Thermal inactivation | 第34页 |
| ·Effect of inhibitors | 第34页 |
| ·Determination of K_m and v_(max) | 第34-35页 |
| ·Iron-sulfur cluster assembly in vitro | 第35-36页 |
| ·Iron-sulfur cluster assembly in apoIscU in vitro | 第35页 |
| ·Iron-sulfur cluster assembly in apoFdx in vitro | 第35-36页 |
| Chapter 3 Expression,purification and mutantion of cysteine desulfurase IscS from A.ferrooxidans | 第36-46页 |
| ·Cloning of the IscS gene from A.ferrooxidans and construction of the mutant plasmids | 第36-37页 |
| ·Expression and purification of IscS wild type and its mutant proteins | 第37-39页 |
| ·UV-visible light scanning of IscS | 第39-41页 |
| ·UV-visible light scanning of IscS | 第39-40页 |
| ·Isolation and analysis of the chromophore present in IscS | 第40-41页 |
| ·Molecular modeling of IscS from A.ferrooxidans | 第41-45页 |
| ·Conclusions | 第45-46页 |
| Chapter 4 Enzymatic Characterization of Cysteine dusulfurase IscS from Acidithiobacillus ferrooxidans | 第46-53页 |
| ·Assay of A.ferrooxidans IscS activity | 第46页 |
| ·Role of mutant sites of IscS for PLP binding and its activity | 第46-47页 |
| ·Enzyme kinetics | 第47-51页 |
| ·Effect of pH and temperature | 第47-49页 |
| ·Thermal inactivation kinetics | 第49页 |
| ·Effect of inhibitors | 第49-51页 |
| ·K_m and v_(max) of IscS from A.ferrooxidans ATCC 23270 | 第51页 |
| ·Conclusions | 第51-53页 |
| Chapter 5 The role of IscS in Iron-sulfur cluster assembly in vitro | 第53-58页 |
| ·Iron-sulfur cluster assembly in vitro requied scaffold proteins | 第53-55页 |
| ·Iron-sulfur cluster assembly in IscU in vitro | 第53页 |
| ·Iron-sulfur cluster assembly in Fdx(Ferredoxin) in vitro | 第53-55页 |
| ·The role of IscS in Iron-sulfur cluster assembly in vitro | 第55-57页 |
| ·The proposed "AUS" model for the iron-sulfur cluster assembly | 第55-56页 |
| ·Inactivation of IscS seriously affected the iron-sulfur cluster assembly | 第56-57页 |
| ·Conclusions | 第57-58页 |
| Chapter 6 Conclusions | 第58-59页 |
| References | 第59-68页 |
| Acknowledgements | 第68-69页 |
| Main achievements | 第69页 |
