臺灣博碩士論文加值系統

在DNA的代謝中，如DNA複製、DNA重組及DNA修復中，單股DNA結合蛋白扮演相當重要的角色。在胃幽門螺旋桿菌中，單股DNA結合蛋白是由ssb基因所轉錄出來的，並且含有179個殘基。我們使用一種截尾突變的胃幽門螺旋菌單股DNA結合蛋白（含有殘基1~134）來和35股的單股DNA形成複合體結晶。我們使用X-ray晶體繞射法來決定此蛋白複合體的結構，最高的解析度到達3.1。胃幽門螺旋菌單股DNA結合蛋白在溶液中是以四聚體的形式存在者，而在蛋白質N端的區域（殘基數1~115）含有一種特殊的蛋白質摺疊區，稱為寡核甘酸結合摺疊區。其它種類的原核生物單股DNA結合蛋白，例如大腸桿菌中，也是含有相同的寡核甘酸結合摺疊區，用來作為單股DNA結合的區域。但是在胃幽門螺旋菌單股DNA結合蛋白複合體晶體結構的研究，我們發現對於單股DNA的結合方式與之前所己知的大腸桿菌單股DNA結合蛋白有很大的不同。胃幽門螺旋菌單股DNA結合蛋白利用疊堆作用力及靜電力來與單股DNA作用。有一些鹼性的殘基，例如精胺酸10、精胺酸35、半胱胺酸108形成相當明顯的鹼性區來與單股DNA作用，另外有兩個芳香性的胺基酸－苯丙胺酸50及色胺酸84以疊堆作用的方法來作用。而由於殘基數116~134的區域較動態無法固定，因此我們仍無法確定其3D結構。而在單股DNA結合蛋白的C端，含有相當酸性的尾端，而被推論用來作為蛋白質-蛋白質之間的交互作用，而此C端作用並且可能可以引發其它結合的蛋白質活性。

Single-stranded DNA binding protein (SSB) plays an important role in DNA metabolism, such as DNA replication, repair, and recombination. SSB of Helicobacter pylori (HpSSB) is encoded by the ssb gene and contains 179 residues. The crystal structure of truncated HpSSB protein (residue 1-134) complexed with dT(pT)34 was determined at 3.1  resolution by X-ray crystallographic method. HpSSB exists as a tetramer in both crystal and solution states. The N-terminal domain (residue 1-115) contains an OB-fold (oligonucleotides binding fold), which is similar with other species like E. coli, to function as an ssDNA binding domain. However, the ssDNA binding mode of tmHpSSB134 exhibits a considerable variability with comparison to that of E. coli. In the structure of tmHpSSB134-dT(pT)34 complex, the ssDNA wraps on the OB-fold with mainly electrostatic and stacking interactions. Several basic residues, Arg10, Arg35, and Lsy108, on the surface of tmHpSSB134 form a significant patch to accommodate the ssDNA binding. Furthermore, two aromatic residues, Phe50 and Trp84, interact with thymidine by stacking interaction. The structure of residues 116-134 was unable to be determined because of its flexibility. Many evidences reveal that the acidic C-terminal tail of SSB might participate in the protein-protein interaction. The C-terminal interactions may trigger the activities of the associated proteins in DNA metabolism.

Chapter 1 Introduction
1.1 Helicobacter pylori
1.2 The target gene hp1245
1.3 Characteristics of E. coli SSB
1.4 The EcoSSB-ssDNA complex
1.5 The associated proteins of SSB
1.6 Structures of single-stranded DNA binding proteins
Chapter 2 Material and Methods
2.1 Cloning, protein expression and purification
2.2 HpSSB-oligonucleotides complex formation
2.3 Analytical gel filtration chromatography
2.4 Analytical ultracentrifugation
2.5 Crystallization
2.6 Data collection and processing
2.7 Structure determination and refinement
Chapter 3 Results and Discussion
3.1 Protein purification and identification
3.1.1 Full-length HpSSB protein
3.1.2 Truncated-mutation HpSSB protein (tmHpSSB134)
3.2 HpSSB-dT(pT)34 complex
3.2.1 Full-length HpSSB-dT(pT)34 complex
3.2.2 tmHpSSB134-dT(pT)34 complex
3.3 Protein crystals
3.4 Space group determination
3.5 The structural phase
3.6 Model building
3.7 Structure refinement
3.8 Structure of tmHpSSB134 protein monomer
3.9 Structure of tmHpSSB134 protein tetramer
3.10 dT(pT)34 conformation
3.11 Residues involved in ssDNA binding
3.12 Crystal packing in tmHpSSB134-dT(pT)34 complex
Chapter 4 Discussion
4.1 Different ssDNA binding mode between HpSSB and EcoSSB
4.2 Potential electrostatic surface of SSBs
4.3 The linkage of ssDNA between SSBs
Figures and Tables
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