跳到主要內容

臺灣博碩士論文加值系統

(3.235.120.150) 您好!臺灣時間:2021/07/31 15:02
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

: 
twitterline
研究生:林張聖彙
研究生(外文):Sheng-HueiLinchung
論文名稱:金屬離子對A群鏈球菌氧化壓力調節因子PerR結構與功能調控之探討
論文名稱(外文):Effect of metal ions on the structure and function of PerR in group A streptococcus
指導教授:王淑鶯
指導教授(外文):Shu-Ying Wang
學位類別:碩士
校院名稱:國立成功大學
系所名稱:微生物及免疫學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:英文
論文頁數:64
中文關鍵詞:A群鏈球菌氧化壓力氧化壓力調控因子PerR金屬
外文關鍵詞:Streptococcus pyogenesGASPerRROS stressmetal
相關次數:
  • 被引用被引用:0
  • 點閱點閱:138
  • 評分評分:
  • 下載下載:5
  • 收藏至我的研究室書目清單書目收藏:0
A群鏈球菌(GAS, Streptococcus pyogenes)為常見引起人類重要疾病的革蘭氏陽性致病菌。A群鏈球菌雖然不具有可以水解過氧化物的觸酶(catalase),卻可以在有氧的環境下生存。目前已知A群鏈球菌有一套精密的機制適應氧化壓力,其中調控因子PerR藉由調控基因的表現來維持菌體內金屬離子的平衡,進一步調節過多的氧化壓力。為了探討PerR調控的分子機制,實驗室先前透過結晶學的方法解出具有結合DNA能力的PerR結構. 結果顯示PerR是同型二聚體 (homodimer),每一單體中包含兩個鋅金屬,一個結合在結構形成區(structural site);另一個結合在調節區 (regulatory site)。參考同源蛋白質B. subtilis PerR的研究,我們認為是否有金屬結合在調節區會決定PerR的結構和功能。為了獲得調節區不含金屬的PerR,我們利用金屬離子有限的M9 medium來表現PerR蛋白質(M9 PerR),並在ICP-OES的分析下證實M9 PerR的金屬含量較先前在LB broth表現的LB PerR低,更進一步發現M9 PerR不具有DNA結合能力。在回填鋅金屬的實驗中,首先觀察到M9 PerR恢復其結合DNA的能力,接著透過測量變性溫度的熱示差掃瞄卡量計 (differential scanning calorimetry, DSC),證明回填鋅金屬可提升蛋白質的穩定性。另一方面,研究調節區帶有H99A突變的PerR蛋白質特性,同樣證實金屬結合在離子調節區決定PerR蛋白質的功能與對金屬的親合力。以上實驗結果證明PerR和鋅金屬結合與否會改變蛋白質的結構,進而決定PerR對DNA結合的能力。
Group A streptococcus (GAS, Streptococcus pyogenes), an important Gram-positive human pathogen, is responsible for a wide spectrum of human diseases which ranges from minor to lethal. GAS is a catalase-deficient bacterium, but it can survive in an oxidative environment. Previous studies have reported that GAS regulates oxidative stress and metal homeostasis through peroxide response regulator, PerR. However, the molecular mechanism of PerR regulation is not clear. The crystal structure of PerR which can bind DNA has been solved. PerR structure is a homodimer with a zinc ion binding to structural and regulatory sites in each monomer. It was speculated that binding of metals to the regulatory site decides PerR function. To test the hypothesis, we purified PerR protein without metal-binding to regulatory site and examined its function and structure. In this study, we overexpressed PerR in metal-limited M9 medium (M9 PerR) and obtained a protein with less metal content compared with LB broth-expressed PerR (LB PerR). M9 PerR showed no DNA-binding ability to dpr promoter on EMSA gel. The binding ability was restored in the presence of various concentrations of zinc ion. Addition of metal ions enhanced M9 PerR protein stability as judged by the metling temperature in the differential scanning calorimetry (DSC) experiments. Characterization of mutant H99A further confirmed the critical role of regulatory site in functional PerR. Based on these findings, we have demonstrated that zinc ions binding to GAS PerR determines protein stability and DNA-binding function.
Chinese Abstract I
Abstract II
Acknowledgment III
Table of contents IV
List of tables VI
List of figures VII
Abbreviation VIII
Chapter 1 Introduction 1
1.1 Clinical significance of Streptococcus pyogenes 1
1.2 Defense mechanism of oxidative stress in Streptococcus pyogenes 2
1.3 Role of PerR in oxidative stress 3
1.4 Molecular mechanism of PerR regulation in Bacillus subtilis 4
1.5 Specific aim 5
Chapter 2 Materials and Methods 6
2.1. Materials 6
2.1.1. Competent cell 6
2.1.2. Primers 6
2.1.3. Plasmids 7
2.1.4. Enzymes 7
2.1.5. Chemicals or reagents 7
2.1.6. Buffers and solutions 9
2.1.7. Kits 11
2.1.8. Instruments 11
2.2 Construction of PerR and PerR mutant 12
2.2.1 Construction of PerR 12
2.2.2 Construction of PerR mutant H99A 12
2.3 Protein overexpression 13
2.3.1 M9 PerR protein overexpression 13
2.3.2 PerR matant H99A protein overexpression 13
2.4 Protein purification 13
2.4.1 M9 PerR protein purification 13
2.4.2 His-tagged H99A PerR protein purification 14
2.5 Gel-filtration analysis 15
2.6 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) 15
2.7. Electrophoretic mobility shift assays (EMSA) 15
2.7.1 Preparation of dpr promoter DNA 15
2.7.2 Mobility shift of M9 PerR and zinc restored M9 PerR 16
2.8 Circular dichroism (CD) 16
2.9 Inductively coupled plasma optical emission spectrometry (ICP-OES) 16
2.10 Differential scanning calorimetry (DSC) 17
2.11 Dynamic light scattering (DLS) 19
2.12 Fourier transform infrared spectroscopy (FT-IR) 19
2.13 Small angle x-ray scattring (SAXS) 20
2.13.1 Sample preparation of M9 PerR for small-angle x-ray scattering 20
2.13.2 SAXS data collection of M9 PerR 20
2.13.3 SAXS data analysis of M9 PerR 21
Chapter 3 Results 22
3.1 Expression and purification of S. pyogenes PerR from M9 medium 22
3.2 Biochemical characterization of M9 PerR 22
3.2.1 M9 PerR is a dimeric protein 22
3.2.2 M9 PerR exhibits impaired binding ability to dpr promoter 23
3.2.3 Secondary structure of PerR by CD 23
3.2.4 Metal content of PerR by ICP-OES 23
3.3 DNA binding ability of M9 PerR is restored by addition of zinc ions 24
3.4 Preincubation of zinc ions increase M9 PerR stability 24
3.5 Zinc ions alter secondary structure composition of M9 PerR 25
3.6 Vibration mode of histidine change after chelating with zinc ions 25
3.7 His99 is critical for PerR DNA-binding and regulatory metal co-ordination 26
3.8 M9 PerR shows partially unfolded on SAXS data analysis 26
Chapter 4 Discussion 28
Chapter 5 Conclusion 31
References 32
Tables 37
Figures 41


Bergmans, H. E., I. M. van Die & W. P. Hoekstra, (1981) Transformation in Escherichia coli: stages in the process. Journal of bacteriology 146: 564-570.
Brenot, A., K. Y. King & M. G. Caparon, (2005) The PerR regulon in peroxide resistance and virulence of Streptococcus pyogenes. Molecular microbiology 55: 221-234.
Brenot, A., K. Y. King, B. Janowiak, O. Griffith & M. G. Caparon, (2004) Contribution of glutathione peroxidase to the virulence of Streptococcus pyogenes. Infection and immunity 72: 408-413.
Brenot, A., B. F. Weston & M. G. Caparon, (2007) A PerR-regulated metal transporter (PmtA) is an interface between oxidative stress and metal homeostasis in Streptococcus pyogenes. Molecular microbiology 63: 1185-1196.
Bruylants, G., J. Wouters & C. Michaux, (2005) Differential scanning calorimetry in life science: thermodynamics, stability, molecular recognition and application in drug design. Current medicinal chemistry 12: 2011-2020.
Carapetis, J. R., A. C. Steer, E. K. Mulholland & M. Weber, (2005) The global burden of group A streptococcal diseases. The Lancet infectious diseases 5: 685-694.
Chao, S.-Y, (2010) Structural studies of the stress response regulator PerR from Streptococcus pyogenes. In Department of Microbiology and Immunology, National Cheng Kung University, Tainan, Taiwan.
Chen, L., L. Keramati & J. D. Helmann, (1995) Coordinate regulation of Bacillus subtilis peroxide stress genes by hydrogen peroxide and metal ions. Proceedings of the National Academy of Sciences of the United States of America 92: 8190-8194.
Chiu, H.-S, (2011) Structural studies of the Streptococcus pyogenes PerR by solution small-angle x-ray scattering. In Department of Microbiology and Immunology, National Cheng Kung University, Tainan, Taiwan.
Cole, J. N., T. C. Barnett, V. Nizet & M. J. Walker, (2011) Molecular insight into invasive group A streptococcal disease. Nature reviews. Microbiology 9: 724-736.
Cornelis, P., Q. Wei, S. C. Andrews & T. Vinckx, (2011) Iron homeostasis and management of oxidative stress response in bacteria. Metallomics : integrated biometal science 3: 540-549.
Duarte, V. & J. M. Latour, (2010) PerR vs OhrR: selective peroxide sensing in Bacillus subtilis. Molecular bioSystems 6: 316-323.
Farr, S. B. & T. Kogoma, (1991) Oxidative stress responses in Escherichia coli and Salmonella typhimurium. Microbiological reviews 55: 561-585.
Faulkner, M. J., Z. Ma, M. Fuangthong & J. D. Helmann, (2012) Derepression of the Bacillus subtilis PerR peroxide stress response leads to iron deficiency. Journal of bacteriology 194: 1226-1235.
Gerlach, D., W. Reichardt & S. Vettermann, (1998) Extracellular superoxide dismutase from Streptococcus pyogenes type 12 strain is manganese-dependent. FEMS microbiology letters 160: 217-224.
Gibson, C. M. & M. G. Caparon, (1996) Insertional inactivation of Streptococcus pyogenes sod suggests that prtF is regulated in response to a superoxide signal. Journal of bacteriology 178: 4688-4695.
Gibson, C. M., T. C. Mallett, A. Claiborne & M. G. Caparon, (2000) Contribution of NADH oxidase to aerobic metabolism of Streptococcus pyogenes. Journal of bacteriology 182: 448-455.
Giedroc, D. P., (2009) Hydrogen peroxide sensing in Bacillus subtilis: it is all about the (metallo) regulator. Molecular microbiology 73: 1-4.
Gill, P., T. T. Moghadam & B. Ranjbar, (2010) Differential scanning calorimetry techniques: applications in biology and nanoscience. Journal of biomolecular techniques : JBT 21: 167-193.
Grifantini, R., C. Toukoki, A. Colaprico & I. Gryllos, (2011) Peroxide stimulon and role of PerR in group A Streptococcus. Journal of bacteriology 193: 6539-6551.
Gryllos, I., R. Grifantini, A. Colaprico, M. E. Cary, A. Hakansson, D. W. Carey, M. Suarez-Chavez, L. A. Kalish, P. D. Mitchell, G. L. White & M. R. Wessels, (2008) PerR confers phagocytic killing resistance and allows pharyngeal colonization by group A Streptococcus. PLoS pathogens 4: e1000145.
Herbig, A. F. & 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. Molecular microbiology 41: 849-859.
Imlay, J. A., (2003) Pathways of oxidative damage. Annual review of microbiology 57: 395-418.
Jacquamet, L., D. A. Traore, J. L. Ferrer, O. Proux, D. Testemale, J. L. Hazemann, E. Nazarenko, A. El Ghazouani, C. Caux-Thang, V. Duarte & J. M. Latour, (2009) Structural characterization of the active form of PerR: insights into the metal-induced activation of PerR and Fur proteins for DNA binding. Molecular microbiology 73: 20-31.
King, K. Y., J. A. Horenstein & M. G. Caparon, (2000) Aerotolerance and peroxide resistance in peroxidase and PerR mutants of Streptococcus pyogenes. Journal of bacteriology 182: 5290-5299.
Konarev, P. V., V. V. Volkov, A. V. Sokolova, M. H. J. Koch & D. I. Svergun, (2003) PRIMUS: a Windows PC-based system for small-angle scattering data analysis. J Appl Crystallogr 36: 1277-1282.
Kong, J. & S. Yu, (2007) Fourier transform infrared spectroscopic analysis of protein secondary structures. Acta biochimica et biophysica Sinica 39: 549-559.
Lee, J. W. & J. D. Helmann, (2006a) Biochemical characterization of the structural Zn2+ site in the Bacillus subtilis peroxide sensor PerR. The Journal of biological chemistry 281: 23567-23578.
Lee, J. W. & J. D. Helmann, (2006b) The PerR transcription factor senses H2O2 by metal-catalysed histidine oxidation. Nature 440: 363-367.
Lee, J. W. & J. D. Helmann, (2007) Functional specialization within the Fur family of metalloregulators. Biometals : an international journal on the role of metal ions in biology, biochemistry, and medicine 20: 485-499.
Lennon, D., (2004) Acute rheumatic fever in children: recognition and treatment. Paediatric drugs 6: 363-373.
Mertens, H. D. & D. I. Svergun, (2010) Structural characterization of proteins and complexes using small-angle X-ray solution scattering. Journal of structural biology 172: 128-141.
Milanino, R., M. Marrella, R. Gasperini, M. Pasqualicchio & G. Velo, (1993) Copper and zinc body levels in inflammation: an overview of the data obtained from animal and human studies. Agents and actions 39: 195-209.
Miller, R. A. & B. E. Britigan, (1997) Role of oxidants in microbial pathophysiology. Clinical microbiology reviews 10: 1-18.
Nobbmann, U., M. Connah, B. Fish, P. Varley, C. Gee, S. Mulot, J. Chen, L. Zhou, Y. Lu, F. Shen, J. Yi & S. E. Harding, (2007) Dynamic light scattering as a relative tool for assessing the molecular integrity and stability of monoclonal antibodies. Biotechnology & genetic engineering reviews 24: 117-128.
O'Brien, K. L., B. Beall, N. L. Barrett, P. R. Cieslak, A. Reingold, M. M. Farley, R. Danila, E. R. Zell, R. Facklam, B. Schwartz & A. Schuchat, (2002) Epidemiology of invasive group A streptococcus disease in the United States, 1995-1999. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America 35: 268-276.
O'Loughlin, R. E., A. Roberson, P. R. Cieslak, R. Lynfield, K. Gershman, A. Craig, B. A. Albanese, M. M. Farley, N. L. Barrett, N. L. Spina, B. Beall, L. H. Harrison, A. Reingold & C. Van Beneden, (2007) The epidemiology of invasive group A streptococcal infection and potential vaccine implications: United States, 2000-2004. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America 45: 853-862.
Perez-Iratxeta, C. & M. A. Andrade-Navarro, (2008) K2D2: estimation of protein secondary structure from circular dichroism spectra. BMC structural biology 8: 25.
Petoukhov, M. V., P. V. Konarev, A. G. Kikhney & D. I. Svergun, (2007) ATSAS 2.1 - towards automated and web-supported small-angle scattering data analysis. J Appl Crystallogr 40: S223-S228.
Ricci, S., R. Janulczyk & L. Bjorck, (2002) The regulator PerR is involved in oxidative stress response and iron homeostasis and is necessary for full virulence of Streptococcus pyogenes. Infection and immunity 70: 4968-4976.
Storz, G. & J. A. Imlay, (1999) Oxidative stress. Current opinion in microbiology 2: 188-194.
Svergun, D. I., (1992) Determination of the Regularization Parameter in Indirect-Transform Methods Using Perceptual Criteria. J Appl Crystallogr 25: 495-503.
Thurnham, D. I., A. S. Mburu, D. L. Mwaniki & A. De Wagt, (2005) Micronutrients in childhood and the influence of subclinical inflammation. The Proceedings of the Nutrition Society 64: 502-509.
Traore, D. A., A. El Ghazouani, S. Ilango, J. Dupuy, L. Jacquamet, J. L. Ferrer, C. Caux-Thang, V. Duarte & J. M. Latour, (2006) Crystal structure of the apo-PerR-Zn protein from Bacillus subtilis. Molecular microbiology 61: 1211-1219.
Traore, D. A., A. El Ghazouani, L. Jacquamet, F. Borel, J. L. Ferrer, D. Lascoux, J. L. Ravanat, M. Jaquinod, G. Blondin, C. Caux-Thang, V. Duarte & J. M. Latour, (2009) Structural and functional characterization of 2-oxo-histidine in oxidized PerR protein. Nature chemical biology 5: 53-59.
Tsou, C. C., C. Chiang-Ni, Y. S. Lin, W. J. Chuang, M. T. Lin, C. C. Liu & J. J. Wu, (2008) An iron-binding protein, Dpr, decreases hydrogen peroxide stress and protects Streptococcus pyogenes against multiple stresses. Infection and immunity 76: 4038-4045.
Tsou, C. C., C. Chiang-Ni, Y. S. Lin, W. J. Chuang, M. T. Lin, C. C. Liu & J. J. Wu, (2010) Oxidative stress and metal ions regulate a ferritin-like gene, dpr, in Streptococcus pyogenes. International journal of medical microbiology : IJMM 300: 259-264.
Versieck, J., (1985) Trace elements in human body fluids and tissues. Critical reviews in clinical laboratory sciences 22: 97-184.

連結至畢業學校之論文網頁點我開啟連結
註: 此連結為研究生畢業學校所提供,不一定有電子全文可供下載,若連結有誤,請點選上方之〝勘誤回報〞功能,我們會盡快修正,謝謝!
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
無相關期刊