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研究生：	費俊憲 Fei, Chun-Hsien
論文名稱：	化膿性鏈球菌PerR與DNA交互作用之研究 Studies on the interactions between the Peroxide Regulator PerR and its cognate DNAin Streptococcus pyogenes
指導教授：	王淑鶯 Wang, Shu-Ying
學位類別：	碩士 Master
系所名稱：	醫學院 - 微生物及免疫學研究所 Department of Microbiology & Immunology
論文出版年：	2012
畢業學年度：	100
語文別：	英文
論文頁數：	58
中文關鍵詞：	化膿性鏈球菌、PerR 、小角度X光散射、晶體結構、BIAcore3000 、平衡解離常數
外文關鍵詞：	Streptococcus pyogenes, PerR, SAXS, crystal structure, BIAcore3000, equilibrium dissociation rate constant (KD)
相關次數：	點閱：144 下載：3
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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

                                    

Bagg, A. and J. B. Neilands (1987). "Ferric uptake regulation protein acts as a repressor, employing iron (II) as a cofactor to bind the operator of an iron transport operon in Escherichia coli." Biochemistry 26(17): 5471-5477.

Bernado, P., E. Mylonas, et al. (2007). "Structural characterization of flexible proteins using small-angle X-ray scattering." J Am Chem Soc 129(17): 5656-5664.

Bessen, D. E. (2009). "Population biology of the human restricted pathogen, Streptococcus pyogenes." Infect Genet Evol 9(4): 581-593.

Bhakdi, S., J. Tranum-Jensen, et al. (1985). "Mechanism of membrane damage by streptolysin-O." Infect Immun 47(1): 52-60.

Brennan, R. G. (1993). "The winged-helix DNA-binding motif: another helix-turn-helix takeoff." Cell 74(5): 773-776.

Brenot, A., K. Y. King, et al. (2005). "The PerR regulon in peroxide resistance and virulence of Streptococcus pyogenes." Mol Microbiol 55(1): 221-234.

Brenot, A., B. F. Weston, et al. (2007). "A PerR-regulated metal transporter (PmtA) is an interface between oxidative stress and metal homeostasis in Streptococcus pyogenes." Mol Microbiol 63(4): 1185-1196.

Bsat, N. and J. D. Helmann (1999). "Interaction of Bacillus subtilis Fur (ferric uptake repressor) with the dhb operator in vitro and in vivo." J Bacteriol 181(14): 4299-4307.

Chao, S. Y. (2010). "Structural studies of the stress response regulator PerR from Streptococcus pyogenes.".

Chen, L., L. P. James, et al. (1993). "Metalloregulation in Bacillus subtilis: isolation and characterization of two genes differentially repressed by metal ions." J Bacteriol 175(17): 5428-5437.

Chen, L., L. Keramati, et al. (1995). "Coordinate regulation of Bacillus subtilis peroxide stress genes by hydrogen peroxide and metal ions." Proc Natl Acad Sci U S A 92(18): 8190-8194.

Chiang-Ni, C. and J. J. Wu (2008). "Effects of streptococcal pyrogenic exotoxin B on pathogenesis of Streptococcus pyogenes." J Formos Med Assoc 107(9): 677-685.

Chiou, H. S. (2011). "Structural studies of the Streptococcus pyogenes PerR by solution small-angle X-ray scattering."

Cunningham, L., M. J. Gruer, et al. (1997). "Transcriptional regulation of the aconitase genes (acnA and acnB) of Escherichia coli." Microbiology. 143(Pt 12): 3795-3805.

Dalton, T. L. and J. R. Scott (2004). "CovS inactivates CovR and is required for growth under conditions of general stress in Streptococcus pyogenes." J Bacteriol 186(12): 3928-3937.

Delany, I., R. Rappuoli, et al. (2004). "Fur functions as an activator and as a repressor of putative virulence genes in Neisseria meningitidis." Molecular Microbiology 52(4): 1081-1090.

Delany, I., G. Spohn, et al. (2001). "The Fur repressor controls transcription of iron-activated and -repressed genes in Helicobacter pylori." Molecular Microbiology 42(5): 1297-1309.

Friedman, Y. E. and M. R. O'Brian (2004). "The ferric uptake regulator (Fur) protein from Bradyrhizobium japonicum is an iron-responsive transcriptional repressor in vitro." J Biol Chem 279(31): 32100-32105.

Fuangthong, M., A. F. Herbig, et al. (2002). "Regulation of the Bacillus subtilis fur and perR genes by PerR: not all members of the PerR regulon are peroxide inducible." J Bacteriol 184(12): 3276-3286.

Gajiwala, K. S. and S. K. Burley (2000). "Winged helix proteins." Curr Opin Struct Biol 10(1): 110-116.

Giedroc, D. P. (2009). "Hydrogen peroxide sensing in Bacillus subtilis: it is all about the (metallo)regulator." Mol Microbiol 73(1): 1-4.
Grifantini, R., C. Toukoki, et al. (2011). "Peroxide stimulon and role of PerR in group A streptococcus." J Bacteriol 193(23): 6539-6551.

Gryllos, I., R. Grifantini, et al. (2008). "PerR confers phagocytic killing resistance and allows pharyngeal colonization by group a streptococcus." Plos Pathogens 4(9).

Hakansson, A., C. C. Bentley, et al. (2005). "Cytolysin-dependent evasion of lysosomal killing." Proc Natl Acad Sci U S A 102(14): 5192-5197.

Herbig, A. F. and J. D. Helmann (2001). "Roles of metal ions and hydrogen peroxide in modulating the interaction of the Bacillus subtilis PerR peroxide regulon repressor with operator DNA." Mol Microbiol 41(4): 849-859.

Horstmann, R. D., H. J. Sievertsen, et al. (1992). "Role of fibrinogen in complement inhibition by streptococcal M protein." Infect Immun 60(12): 5036-5041.

Huffman, J. L. and R. G. Brennan (2002). "Prokaryotic transcription regulators: more than just the helix-turn-helix motif." Curr Opin Struct Biol 12(1): 98-106.

Jacquamet, L., D. A. Traore, et al. (2009). "Structural characterization of the active form of PerR: insights into the metal-induced activation of PerR and Fur proteins for DNA binding." Mol Microbiol 73(1): 20-31.

King, K. Y., J. A. Horenstein, et al. (2000). "Aerotolerance and peroxide resistance in peroxidase and PerR mutants of Streptococcus pyogenes." J Bacteriol 182(19): 5290-5299.

Kozin, M. B. and D. I. Svergun "Automated matching of high- and low-resolution structural models."

Kuo, C. F., Y. S. Lin, et al. (2008). "Degradation of complement 3 by streptococcal pyrogenic exotoxin B inhibits complement activation and neutrophil opsonophagocytosis." Infect Immun 76(3): 1163-1169.

Lamagni, T. L., J. Darenberg, et al. (2008). "Epidemiology of severe Streptococcus pyogenes disease in Europe." J Clin Microbiol 46(7): 2359-2367.

Lamagni, T. L., A. Efstratiou, et al. (2005). "The epidemiology of severe Streptococcus pyogenes associated disease in Europe." Euro Surveill 10(9): 179-184.

Lancefield, R. C. (1962). "Current knowledge of type-specific M antigens of group A streptococci." J Immunol 89: 307-313.

Lewin, A. C., P. A. Doughty, et al. (2002). "The ferric uptake regulator of Pseudomonas aeruginosa has no essential cysteine residues and does not contain a structural zinc ion." Microbiology 148(Pt 8): 2449-2456.

Li, J. M., Y. L. Su, et al. (2011). "Molecular characterization and oxidative stress response of an intracellular Cu/Zn superoxide dismutase (CuZnSOD) of the whitefly, Bemisia tabaci." Arch Insect Biochem Physiol 3(10): 20428.

Ligeza, A., A. N. Tikhonov, et al. (1998). "Oxygen permeability of thylakoid membranes: electron paramagnetic resonance spin labeling study." Biochim Biophys Acta. 1365(3): 453-463.

Liochev, S. I. and I. Fridovich (1992). "Fumarase C, the stable fumarase of Escherichia coli, is controlled by the soxRS regulon." Proc Natl Acad Sci U S A. 89(13): 5892-5896.

Luscombe, N. M., R. A. Laskowski, et al. (2001). "Amino acid-base interactions: a three-dimensional analysis of protein-DNA interactions at an atomic level." Nucleic Acids Res 29(13): 2860-2874.

Mongkolsuk, S. and J. D. Helmann (2002). "Regulation of inducible peroxide stress responses." Mol Microbiol 45(1): 9-15.

Myszka, D. G. (1999). "Survey of the 1998 optical biosensor literature." J Mol Recognit 12(6): 390-408.

Okada, N., M. K. Liszewski, et al. (1995). "Membrane cofactor protein (CD46) is a keratinocyte receptor for the M protein of the group A streptococcus." Proc Natl Acad Sci U S A 92(7): 2489-2493.

P. V. Konarev, M. V. P. V. V. V. and D. I. Svergun "ATSAS 2.1, a program package for small-angle scattering data analysis."
Ricci, S., R. Janulczyk, et al. (2002). "The regulator PerR is involved in oxidative stress response and iron homeostasis and is necessary for full virulence of Streptococcus pyogenes." Infect Immun 70(9): 4968-4976.

Robinson, J. M. (2008). "Reactive oxygen species in phagocytic leukocytes." Histochem Cell Biol 130(2): 281-297.

Safo, M. K., Q. Zhao, et al. (2005). "Crystal structures of the BlaI repressor from Staphylococcus aureus and its complex with DNA: insights into transcriptional regulation of the bla and mec operons." J Bacteriol 187(5): 1833-1844.

Schneidman-Duhovny, D., M. Hammel, et al. (2011). "Macromolecular docking restrained by a small angle X-ray scattering profile." J Struct Biol 173(3): 461-471.

Smith, J. L. (2004). "The physiological role of ferritin-like compounds in bacteria." Crit Rev Microbiol 30(3): 173-185.

Traore, D. A., A. El Ghazouani, et al. (2006). "Crystal structure of the apo-PerR-Zn protein from Bacillus subtilis." Mol Microbiol 61(5): 1211-1219.

Traore, D. A., A. El Ghazouani, et al. (2009). "Structural and functional characterization of 2-oxo-histidine in oxidized PerR protein." Nat Chem Biol 5(1): 53-59.

Tsou, C. C. (2010). "The roles of dpr in anti-oxidative and -metal stresses and its regulation by PerR in group A streptococcus."

Tsou, C. C., C. Chiang-Ni, et al. (2008). "An iron-binding protein, Dpr, decreases hydrogen peroxide stress and protects Streptococcus pyogenes against multiple stresses." Infect Immun 76(9): 4038-4045.

Tsou, C. C., C. Chiang-Ni, et al. (2010). "Oxidative stress and metal ions regulate a ferritin-like gene, dpr, in Streptococcus pyogenes." Int J Med Microbiol 300(4): 259-264.

Volkov, V. V. and D. I. Svergun "Uniqueness of ab initio shape determination in small-angle scattering."

Wang, S., R. T. Fleming, et al. (2006). "Structure of the Escherichia coli FlhDC complex, a prokaryotic heteromeric regulator of transcription." J Mol Biol 355(4): 798-808.

Wen, Y. T., C. C. Tsou, et al. (2011). "Differential secretomics of Streptococcus pyogenes reveals a novel peroxide regulator (PerR)-regulated extracellular virulence factor mitogen factor 3 (MF3)." Mol Cell Proteomics 10(9): M110 007013.

Wolberger, C. and R. Campbell (2000). "New perch for the winged helix." Nat Struct Biol 7(4): 261-262.

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