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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 詹迺立(Nei-Li Chan) | |
dc.contributor.author | Ching-Hui Lai | en |
dc.contributor.author | 賴青慧 | zh_TW |
dc.date.accessioned | 2021-06-15T11:30:45Z | - |
dc.date.available | 2018-08-26 | |
dc.date.copyright | 2016-08-26 | |
dc.date.issued | 2016 | |
dc.date.submitted | 2016-08-17 | |
dc.identifier.citation | 1 Jarup, L. Hazards of heavy metal contamination. Br Med Bull 68, 167-182 (2003).
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Heavy Metals in the Environment: Origin, Interaction and Remediation. Vol. 6 (London: Academic Press, 2002). 9 Zhao, M., Xu, Y., Zhang, C., Rong, H. & Zeng, G. New trends in removing heavy metals from wastewater. Appl Microbiol Biotechnol, doi:10.1007/s00253-016-7646-x (2016). 10 Gibb, H. & O'Leary, K. G. Mercury exposure and health impacts among individuals in the artisanal and small-scale gold mining community: a comprehensive review. Environ Health Perspect 122, 667-672, doi:10.1289/ehp.1307864 (2014). 11 Harada, M. Minamata disease: methylmercury poisoning in Japan caused by environmental pollution. Crit Rev Toxicol 25, 1-24, doi:10.3109/10408449509089885 (1995). 12 Park, J. D. & Zheng, W. Human exposure and health effects of inorganic and elemental mercury. J Prev Med Public Health 45, 344-352, doi:10.3961/jpmph.2012.45.6.344 (2012). 13 Wang, X. & Horisberger, J. D. Mercury binding site on Na+/K(+)-ATPase: a cysteine in the first transmembrane segment. 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Genetic analysis of the Tn21 mer operator-promoter. J Bacteriol 174, 2160-2171 (1992). 21 Summers, A. O. Untwist and shout: a heavy metal-responsive transcriptional regulator. J Bacteriol 174, 3097-3101 (1992). 22 Gutknecht, J. Inorganic mercury (Hg2+) transport through lipid bilayer membranes. The Journal of Membrane Biology 61, 61-66, doi:10.1007/BF01870753 (1981). 23 Barkay, T., Miller, S. M. & Summers, A. O. Bacterial mercury resistance from atoms to ecosystems. FEMS Microbiol Rev 27, 355-384 (2003). 24 Mukhopadhyay, D., Yu, H. R., Nucifora, G. & Misra, T. K. Purification and functional characterization of MerD. A coregulator of the mercury resistance operon in gram-negative bacteria. J Biol Chem 266, 18538-18542 (1991). 25 Chen, C. Y., Hsieh, J. L., Silver, S., Endo, G. & Huang, C. C. Interactions between two MerR regulators and three operator/promoter regions in the mercury resistance module of Bacillus megaterium. Biosci Biotechnol Biochem 72, 2403-2410, doi:10.1271/bbb.80294 (2008). 26 Parkhill, J. & Brown, N. L. Site-specific insertion and deletion mutants in the mer promoter-operator region of Tn501; the nineteen base-pair spacer is essential for normal induction of the promoter by MerR. Nucleic Acids Res 18, 5157-5162 (1990). 27 Heldwein, E. E. & Brennan, R. G. Crystal structure of the transcription activator BmrR bound to DNA and a drug. Nature 409, 378-382, doi:10.1038/35053138 (2001). 28 Watanabe, S., Kita, A., Kobayashi, K. & Miki, K. Crystal structure of the [2Fe-2S] oxidative-stress sensor SoxR bound to DNA. Proc Natl Acad Sci U S A 105, 4121-4126, doi:10.1073/pnas.0709188105 (2008). 29 Philips, S. J. et al. TRANSCRIPTION. Allosteric transcriptional regulation via changes in the overall topology of the core promoter. Science 349, 877-881, doi:10.1126/science.aaa9809 (2015). 30 Chang, C. C., Lin, L. Y., Zou, X. W., Huang, C. C. & Chan, N. L. Structural basis of the mercury(II)-mediated conformational switching of the dual-function transcriptional regulator MerR. Nucleic Acids Res 43, 7612-7623, doi:10.1093/nar/gkv681 (2015). 31 Steele, R. A. & Opella, S. J. Structures of the reduced and mercury-bound forms of MerP, the periplasmic protein from the bacterial mercury detoxification system. Biochemistry 36, 6885-6895, doi:10.1021/bi9631632 (1997). 32 Changela, A. et al. Molecular basis of metal-ion selectivity and zeptomolar sensitivity by CueR. Science 301, 1383-1387, doi:10.1126/science.1085950 (2003). 33 Ralston, D. M. & O'Halloran, T. V. Ultrasensitivity and heavy-metal selectivity of the allosterically modulated MerR transcription complex. Proc Natl Acad Sci U S A 87, 3846-3850 (1990). 34 Ahmed, M., Borsch, C. M., Taylor, S. S., Vazquez-Laslop, N. & Neyfakh, A. A. A protein that activates expression of a multidrug efflux transporter upon binding the transporter substrates. J Biol Chem 269, 28506-28513 (1994). 35 Russo Krauss, I., Merlino, A., Vergara, A. & Sica, F. An overview of biological macromolecule crystallization. Int J Mol Sci 14, 11643-11691, doi:10.3390/ijms140611643 (2013). 36 Walter, T. S. et al. Semi-automated microseeding of nanolitre crystallization experiments. Acta Crystallogr Sect F Struct Biol Cryst Commun 64, 14-18, doi:10.1107/S1744309107057260 (2008). 37 Oswald, C., Smits, S. H., Bremer, E. & Schmitt, L. Microseeding - a powerful tool for crystallizing proteins complexed with hydrolyzable substrates. Int J Mol Sci 9, 1131-1141, doi:10.3390/ijms9071131 (2008). 38 Helmann, J. D., Wang, Y., Mahler, I. & Walsh, C. T. Homologous metalloregulatory proteins from both gram-positive and gram-negative bacteria control transcription of mercury resistance operons. J Bacteriol 171, 222-229 (1989). 39 O'Halloran, T. & Walsh, C. Metalloregulatory DNA-binding protein encoded by the merR gene: isolation and characterization. Science 235, 211-214 (1987). 40 Shewchuk, L. M., Verdine, G. L. & Walsh, C. T. Transcriptional switching by the metalloregulatory MerR protein: initial characterization of DNA and mercury (II) binding activities. Biochemistry 28, 2331-2339 (1989). 41 Kr zel, A. et al. Coordination of heavy metals by dithiothreitol, a commonly used thiol group protectant. J Inorg Biochem 84, 77-88 (2001). | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/49481 | - |
dc.description.abstract | 重金屬以各種形式廣泛存在於大自然中,有些重金屬為生物體維持合成代謝等生理運作所不可或缺,有些重金屬則無明顯之生理功能、但以高毒性著稱,例如:鉛、砷、鎘、汞等,其存在往往導致個體的死亡。為了維持必須微量元素之恆定與抵抗高毒性之重金屬,生物體發展出極為多元的重金屬抗性系統來調控重金屬之濃度。
MerR家族為一系列之轉錄調控蛋白,最典型之蛋白為MerR,可結合mer操縱組調控基因之轉錄,為一個具有雙重功能的蛋白。當環境中缺少誘導物汞離子時,MerR扮演抑制子之角色,然而當汞離子存在時MerR則扮演活化子之角色。一旦與誘導物結合,MerR會藉由本身之構型改變,帶動mer O/P DNA上的兩個RNA聚合酶結合位重新校正排列成適合轉錄之相對位置,使mer operon和RNA聚合酶形成開放式複合體,mer operon上與抵抗汞相關之蛋白得以表現,這些蛋白主要包含:間質蛋白MerP、內膜蛋白MerT及MerC、汞離子還原酶MerA、轉錄調控因子MerR及轉錄調控之共同抑制子MerD。 本實驗室已於先前解出來自於Bacillus megaterium之apo-MerR1及Hg2+-bound MerR1結構,分別對應MerR1之抑制構型及活化構型。為描繪抑制及活化構型MerR1調控mer operon之細節,我需要得到apo-MerR1與merR1 O/P DNA複合體及Hg2+-bound MerR1與merR1 O/P DNA複合體之結構。此外,為解析汞離子與MerR1之三個cysteine結合之順序性、並比較MerR1與同源蛋白CueR結合重金屬方式之異同,本實驗室利用突變型之MerR1與汞離子進行共結晶。我亦嘗試解出MerR1與鋅離子或鎘離子之結構,以解釋MerR1對於汞離子之特異性。 我已在merR1 O/P DNA存在下得到apo-MerR1之晶體,然而經過各種純化、養晶條件的調整,包括:調整鹽類濃度、增加共養晶DNA比例等,在結構中皆未能發現DNA的存在,未來我將嘗試以Pseudomonas aeruginosa (Tn501) MerR進行共結晶實驗。探討MerR與金屬離子交互作用之部分,於MerR1_C123A突變型之晶體結構中,未觀察到汞離子之結合,由於汞離子若非以最理想之三角平面與MerR1進行結合時,彼此間之親和性應會顯著降低,因此之後進行共結晶實驗需盡可能提高汞離子之濃度;於MerR1與鎘離子共結晶的晶體結構中,亦未觀察到鎘離子之電子密度,由於用於純化之還原劑DTT可能與鎘離子產生聚合,極可能對共結晶產生干擾,因此未來進行共結晶時DTT濃度需降低或以其他還原劑置換,汞離子濃度亦需進一步提高。 | zh_TW |
dc.description.abstract | There are a variety of different forms of heavy metals existing in the natural environment. While some of them are essential for metabolic pathways and various cellular processes, higher intracellular levels of heavy metals may lead to cytotoxicity. Therefore, deficiency in the supply and excessive accumulation of essential metals are both life-threatening, and their cellular levels must be carefully maintained. Another group of heavy metals, such as Hg, Pb, As and Cd, are well known for their extreme toxicity. To regulate the homeostasis of essential metals and to guard against the lethal effects of toxic heavy metals, all organisms are equipped with heavy metal resistance systems.
The expression of metal resistance systems is controlled by a group of transcriptional regulatory proteins belonging to the MerR protein family. MerR regulates the expression of genes encoded by the mercury resistance operon (mer operon) by binding to the operator/promoter (O/P) region of the operon. MerR is best known for its dual functionality: it acts as a repressor when Hg2+ is absent, whereas MerR is converted to an activator in the presence of Hg2+. Upon Hg2+ binding, the conformation of MerR is altered to induce a directional twisting of mer O/P DNA, which realigns the two RNA polymerase binding sites to facilitate open complex formation and transcription from the mer operon. Our lab has previously solved the crystal structures of apo-MerR1 and Hg2+-bound MerR1 from Bacillus megaterium, representing respectively the repressor and activator form of MerR. To address how MerR1 regulates the activity of mer operon in greater detail, it is necessary to obtain the structures of apo- and Hg2+-bound MerR1 in complexes with merR1 O/P DNA. In addition, we attempted to address how the Hg2+-binding site with trigonal planar coordination geometry is assembled by producing site-directed MerR1 mutants in which the three Hg2+-ligating cysteine residues are individually mutated. We also attempted to characterize the structures of Zn2+- and Cd2+-bound MerR1 to understand the high specificity of MerR1 towards Hg2. To these ends, we have produced several types of MerR1 crystals in the presence of merR1 O/P DNA. However, no DNA was observed in the resolved structures. We will try to use MerR from Pseudomonas aeruginosa to continue our co-crystallized experiments in the future. We have also crystallized MerR1_C123A mutant in the presence of Hg2, but no electron density of Hg2+ was observed, suggesting that it is necessary to increase the concentration of Hg2+ to compensate the reduced affinity of MerR1 for Hg2+. Likewise, for the MerR1 crystals obtained in the presence of Cd2+, no electron density corresponds to Cd2+ was observed, consistent with its lower affinity towards Cd2+. Given that the thiol-containing reducing agent DTT may interact with Hg2+ and Cd2+, and thus interferes with the co-crystallization, future crystallization trials will be conducted by replacing DTT with other reducing agents. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T11:30:45Z (GMT). No. of bitstreams: 1 ntu-105-R03442030-1.pdf: 3630532 bytes, checksum: 9ae7c8ccac876387f2e978025a62aa7c (MD5) Previous issue date: 2016 | en |
dc.description.tableofcontents | 謝誌 I
摘要 II Abstract IV 縮寫表 VI 目錄 VII 圖目錄 IX 表目錄 X 一、前言 1 1.1. 重金屬的定義與簡介 1 1.2. 細菌排除有害重金屬的方式 2 1.3. mer operone之組成及抵抗汞之機制 3 1.4. MerR1調控merR1 operon之方式 4 1.5. 已知結構──apo-MerR1與Hg2+-bound MerR1 5 1.6. MerR1的三個cysteine與Hg2+結合順序之討論 6 1.7. MerR1對Hg2+特異性的討論 7 1.8. 研究動機 8 二、材料與方法 10 2.1. 蛋白質表現系統 10 2.1.1. Bacillus megaterium之MerR1質體建構與表現蛋白菌株 10 2.1.2. Pseudomonas aeruginosa之MerR1質體建構與表現蛋白菌株 10 2.1.3. MerR之大量表現 10 2.2. 蛋白質純化 11 2.2.1. Bacillus megaterium MerR之純化(包含MerR1之突變型) 11 2.2.2. Pseudomonas aeruginosa MerR之純化 14 2.3. 蛋白質濃縮及定量 15 2.3.1. 蛋白之濃縮 15 2.3.2. 蛋白之定量 16 2.4. 共養晶之DNA 16 2.4.1. 單股核苷酸序列設計 16 2.4.2. 雙股DNA形成 (duplex formation) 16 2.4.3. 凝膠遷移率變動分析 (Gel mobility shift assay) 17 2.5. 蛋白質活性測試 17 2.5.1. 凝膠遷移率變動分析(Gel mobility shift assay) 17 2.6. 蛋白質晶體培養 18 2.6.1. 養晶樣品製備 18 2.6.2. 結晶方法 19 2.6.3. 結晶條件 20 2.7. 蛋白質晶體之X-ray繞射數據收集與結構解析 20 2.7.1. 共養晶晶體之保存與運輸 20 2.7.2. 共養晶晶體之X-光繞射數據收集與結構解析 21 2.8. 定位點突變 (site-directed mutagenesis) 21 三、結果 23 3.1. 革蘭氏陽性菌Bacillus megaterium MerR1與merR1 O/P DNA之交互作用 23 3.1.1. MerR1的表現與純化 23 3.1.2. MerR1的結晶與微晶種 (microseeding)養晶 23 3.1.3. 改變條件與設計新merR1 O/P DNA 24 3.2. 革蘭氏陰性菌Pseudomonas aeruginosa MerR 25 3.2.1. MerR的表現與純化 25 3.3. MerR1突變型與Hg2+之交互作用 25 3.4. MerR1和2B族金屬Zn (II)與Cd (II)之交互作用 26 四、討論 27 4.1. 蛋白質純化 27 4.2. 蛋白質濃縮 27 4.3. 雙股merR1 O/P DNA的形成 (duplex formation) 28 4.4. Hg2+-bound MerR1與merR1 O/P DNA複合體晶體培養 29 4.5. MerR1突變蛋白之結構解析 30 4.6. MerR1與2B族金屬鋅與鎘之交互作用 30 圖 32 表 48 參考文獻 64 | |
dc.language.iso | zh-TW | |
dc.title | 探討MerR蛋白與汞抗性操縱組及金屬離子汞、鎘之交互作用 | zh_TW |
dc.title | Interaction of MerR with mer operon, Hg2+ and Cd2+ | en |
dc.type | Thesis | |
dc.date.schoolyear | 104-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 黃介辰(Chieh-Chen Huang),冀宏源(Hung-Yuan Chi) | |
dc.subject.keyword | MerR家族,mer操縱組,抑制構型,活化構型, | zh_TW |
dc.subject.keyword | MerR family,mer operon,active form,suppress form, | en |
dc.relation.page | 67 | |
dc.identifier.doi | 10.6342/NTU201603079 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2016-08-17 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 生物化學暨分子生物學研究所 | zh_TW |
顯示於系所單位: | 生物化學暨分子生物學科研究所 |
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