臺灣博碩士論文加值系統

中文摘要

單股DNA結合蛋白於DNA代謝，例如DNA複製、修復及重組等過程中扮演重要角色。蛋白質結構上，單股DNA結合蛋白主要包含兩區域，其中N端與單股DNA結合有關區域又稱寡醣核苷酸結合結構區，而C端最末則包含一段保留序列，被認為與其他參與DNA代謝過程相關蛋白質之結合有關。本研究中，我們利用X光晶體繞射實驗並利用分子取代法決定C端截尾之胃幽門螺旋桿菌單股DNA結合蛋白與兩段聚核苷酸 (各包含35個胸腺嘧啶核苷) 所形成復合物之晶體結構，所得晶體最高解析度達2.3 Å。經結構分析，單股DNA主要藉由疏水性及靜電作用力纏繞於蛋白表面，其中與疏水性作用力有關之芳香族胺基酸殘基，包括苯丙胺酸37、50、56及色氨酸84均參與作用，其作用方式即利用其苯環衍生物與核甘酸之鹼基進行堆疊作用。於靜電作用力方面，帶正電荷之鹼性胺基酸殘基，例如精胺酸10、36、54及94，於蛋白表面形成一明顯帶正電區域以提供單股DNA結合。

Abstract

Single-stranded DNA binding protein (SSB) plays an important role in DNA metabolism, such as DNA replication, repair and recombination. The N-terminal domain of SSB forms an oligonucleotides/oligosaccharides binding (OB) fold, which function as single-stranded DNA (ssDNA) binding domain. The C-terminal conserved tail is supposed to participate in the protein-protein interaction. The crystal structure of C-terminal truncated SSB from Helicobacter pylori (residues 1-134, HpSSB134) bound to two dT(pT)34 [HpSSB134-dT(pT)34] was determined by molecular replacement method at 2.3 Å. The ssDNA wraps around four subunits of HpSSB134 by the electrostatic and hydrophobic stacking interactions. Four aromatic residues, Phe37, Phe50, Phe56 and Trp84, interact with the base of ssDNA by stacking arrangement. Meanwhile, four basic residues, Arg10, Arg36, Arg54 and Arg94, on the surface of HpSSB134 form a significant electrostatic path corresponding to the ssDNA binding.

中文摘要 i
Abstract ii
Contents iii
Chapter 1 Introduction 1
1.1 Helicobacter pylori 1
1.2 Single-stranded DNA binding proteins (SSB) 1
1.3 Characteristics of OB-fold in SSB 2
1.4 The binding modes of SSB 3
1.5 The associated proteins of SSB 4
1.6 Other single-stranded DNA binding proteins 4

Chapter 2 Materials and methods 6
2.1 Cloning, protein expression and purification 6
2.2 HpSSB134-dT(pT)34 complex preparation 7
2.3 Analytical gel filtration chromatography 7
2.4 Analytical ultracentrifugation 7
2.5 Crystallization 8
2.6 Data collection and processing 8
2.7 Structure determination and refinement 8

Chapter 3 Results 10
3.1 Protein purification and identification 10
3.1.1 Full-length HpSSB 10
3.1.2 C-terminal truncated HpSSB 10
3.2 HpSSB134-dT(pT)34 complex 11
3.3 Protein crystals 12
3.4 Space group determination 12
3.5 Structural determination 12
3.6 Model building 13
3.7 Structural refinement 13
3.8 Overall Structure of HpSSB134 14
3.9 Quaternary structure of HpSSB134 15
3.10 dT(pT)34 conformation 16
3.11 Residues involved in ssDNA binding 17
3.12 Crystal packing in HpSSB134-dT(pT)34 complex 18

Chapter 4 Discussions 20
4.1 Comparison of ssDNA binding mode between HpSSB134
and EcoSS 20
4.2 Potential electrostatic surface of SSB 20
4.3 The linkage of ssDNA between SSB 21

Appendix Figures and Tables 22

References 53

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