臺灣博碩士論文加值系統

化膿性鏈球菌(Streptococcus pyogenes)，又稱A群鏈球菌(Group A streptococcus, GAS)，是造成許多嚴重人類疾病的格蘭氏陽性菌，例如鏈球菌中毒性休克症候群(streptococcal toxic shock syndrome)和壞死性筋膜炎(necrotizing fasciitis)。A群鏈球菌是一隻過氧化氫酶(catalase)陰性的細菌，然而，在感染過程中卻能夠抵禦來自活性氧分子(ROS)產生的氧化壓力(oxidative stress)。PerR已在過去的研究中被證實是調節過氧化物的重要調控因子。當受到環境中ROS的刺激，PerR便從peroxide-resistance protein (Dpr)的啟動子游離，使dpr基因表現以減少來自ROS造成的傷害。我們致力於探討PerR如何與DNA交互作用。我們實驗室先前已經成功解出PerR的晶體結構。將PerR結構與同樣帶有Winged-helix DNA結合部位的金黃色葡萄球菌BlaI蛋白疊合，我們預測四個residues可能參與和DNA的交互作用之中，分別是Tyr67、Asn68、Lys71和Lys83。本篇研究中，我們建構四個突變蛋白並將其純化至95%純度。分子篩層析法結果顯示所有突變型蛋白都能與野生型(wild-type)相同形成二聚體(dimer)，表示這些突變並不影響蛋白質偶合功能。電泳遷移實驗(electrophoresis mobility shift assay)結果顯示將PerR的Tyr67、Asn68、Lys71和Lys83突變為Ala後降低了PerR結合DNA的能力。我們同時用BIAcore3000系統定量出野生型PerR和N68A突變PerR蛋白結合DNA的平衡解離常數(equilibrium dissociation rate constant, KD)。我們也解決PerR蛋白製備的困難，進行小角度X光散射(SAXS)實驗，收集到較好的SAXS scattering data。另外小角度X光散射實驗指出PerR在長晶過程中會有些微的構型改變，但整體來說，在溶液中具有DNA結合能力的PerR之構型與PerR晶體結構之構型是一致的。在未來，若要進一步在分子層級研究PerR和DNA交互作用，則PerR和DNA的複合物晶體結構會是最直接有力的研究材料。

Streptococcus pyogenes (group A streptococcus, GAS) is a gram-positive bacterium which causes severe human diseases, such as streptococcal toxic shock syndrome and necrotizing fasciitis. GAS is a catalase-negative pathogen; however, it can cope with oxidative stress during infection. Peroxide Regulator (PerR) has been shown to be an important transcription factor involved in peroxide regulation. When stimulated by reactive oxygen species (ROS), PerR dissociates from the promoter of peroxide-resistance protein Dpr, allowing dpr gene expression which in turn reduces the damage caused by ROS. This thesis project is to study the molecular interaction between PerR and its cognate DNA, thus to understand how GAS survives in oxidative environment. The crystal structure of PerR was solved to 1.6 Å in our lab. Superimposition of the wing-helix DNA-binding sites of GAS PerR structure with that of Staphyloccocus aureus BlaI, it suggests that four residues in PerR, Tyr67, Asn68, Lys71 and Lys83 might involve in interaction with DNA. In this study, four mutants (Tyr67Ala, Asn68Ala, Lys71Ala and Lys83Ala) were constructed and purified up to 95% purity. Size exclusion chromatography analysis showed that all four mutants are in dimeric form. Electrophoresis mobility shift assay showed that substitution of the residues Tyr67, Asn68, Lys71 and Lys83 to alanine reduces the DNA binding affinity of PerR. The equilibrium dissociation rate constant (KD) of wild-type PerR and PerR N68A was characterized by BIAcore3000 system. The aggregation problem of the sample preparation for conducting small-angle X-ray scattering (SAXS) was solved and better SAXS data were collected. SAXS results indicate that GAS PerR undergo locally conformational change during crystallization, but the overall conformation of GAS PerR in crystal structure and in solution, which possesses DNA-binding ability, is identical. Further studies of PerR-DNA complex is required to uncover the molecular mechanisms of the biological interaction at atomic level.

中文摘要 I
ABSTRACT III
誌謝 V
ABBREVIATION X
CHAPTER 1 INTRODUCTION 1
1.1 INTRODUCTION OF STREPTOCOCCUS PYOGENES 1
1.2 OXIDATIVE STRESS FOR GAS 2
1.3 THE PEROXIDE REGULON REPRESSOR PERR 3
1.4 PRINCIPLE OF SMALL-ANGLE X-RAY SCATTERING (SAXS) 5
1.5 SAXS DATA ANALYSIS AND PROCESS BY ATSAS 7
1.6 THE AIM OF THIS STUDY 8
CHAPTER 2 EXPERIMENTAL PROCEDURES 9
2.1 MATERIALS 9
2.1.1 Bacteria strains 9
2.1.2 Plasmid 9
2.1.3 Primers 10
2.1.4 Chemicals and other material 10
2.1.5 Buffers 13
2.2 METHODS 17
2.2.1 Construction of S. pyogenes PerR and PerR mutant plasmids 17
2.2.2 Transformation of S. pyogenes PerR plasmids 18
2.2.3 Overexpression of PerR and PerR mutants 19
2.2.4 Purification of PerR and PerR mutants 19
2.2.5 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) 20
2.2.6 Size-exclusion chromatography 20
2.2.7 dpr promoter DNA amplification 20
2.2.8 Electrophoretic mobility shift assays 21
2.2.9 BIAcore 3000 analysis 21
2.2.10 Small-angle X-ray scattering (SAXS) 23
CHAPTER 3 RESULTS 25
3.1 PURIFICATION AND CHARACTERIZATION OF S. PYOGENES PERR AND PERR MUTANTS 25
3.2 MUTATION OF PUTATIVE DNA-BINDING SITE RESIDUES IN PERR REDUCES DNA BINDING AFFINITY 25
3.3 SURFACE PLASMON RESONANCE KINETIC ANALYSIS OF THE PERR-DNA INTERACTION 26
3.4 BINDING OF PERR TO DNA DOES NOT INDUCE BENDING OF DNA 26
3.5 SAXS DATA COLLECTION OF S. PYOGENES PERR 26
3.6 RIGID BODY MODELING OF SAXS DATA 27
CHAPTER 4 DISCUSSION 29
CHAPTER 5 CONCLUSIONS 33
REFERENCES 34
TABLES 40
FIGURES 43

Table List
TABLES 40
TABLE 2.1 LIST OF PRIMER SEQUENCES 40
TABLE 2.2 LIST OF PALINDROMIC DNA SEQUENCES 42

Figure List
FIGURES 43
FIGURE 1.1 CRYSTAL STRUCTURES OF S. PYOGENES AND B. SUBTILIS PERR. 43
FIGURE 1.2 GAS PERR CONTAINS WINGED-HELIX DNA-BINDING DOMAIN 44
FIGURE 1.3 COMPARISON OF DNA-BINDING DOMAIN OF S. PYOGENES PERR WITH S. AUREUS BLAI 45
FIGURE 2.1 PROTEIN SEQUENCE OF S. PYOGENES PERR. 46
FIGURE 3.1 OVEREXPRESSION OF PERR AND PERR MUTANTS. 46
FIGURE 3.2 PURIFICATION OF PERR AND PERR MUTANTS BY NI2+-NTA CHROMATOGRAPHY AND SIZE-EXCLUSION CHROMATAGRAPHY 47
FIGURE 3.3 ELECTROPHORESIS MOBILITY SHIFT ASSAY ANALYSIS OF WILD-TYPE PERR AND MUTANTS. 49
FIGURE 3.4 FITTING CURVES OF WILD-TYPE PERR AND PERR N68A WITH DPR PROMOTER DNA USING 1:1 FITTING MODEL. 50
FIGURE 3.5 ELECTROPHORETIC MOBILITY SHIFT ASSAY SHOWS THAT BINDING OF PERR TO DPR PROMOTER DNA DOES NOT INDUCE DNA BENDING. 51
FIGURE 3.6 SCATTERING CURVES OF S.PYOGENES WILD-TYPE PERR. 52
FIGURE 3.7 GUINIER PLOT, PAIR-DISTRIBUTION FUNCTIONA AND KRATKY PLOT OF WILD-TYPE PERR. 53
FIGURE 3.8 CRYSTALLOGRAPHIC STRUCTURE AND SAXS MODEL OF WILD-TYPE PERR. 54
FIGURE 3.9 COMPARISON OF EXPERIMENTAL CURVE WITH THEORETICAL CURVE BY CRYSOL. 55
FIGURE 3.10 SASREF ANALYSIS OF S. PYOGENES PERR 56
FIGURE 4.1 TOPOLOGY OF WINGED-HELIX MOTIF OF GAS PERR AND S. AUREUS BLAI. 57
FIGURE 4.2 SEQUENCE ALIGNMENT OF S. AUREUS BLAI AND GAS PERR 57
FIGURE 4.3 FOXSDOCK ANALYSIS OF S. PYOGENES PERR 58

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