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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 詹迺立(Nei-Li Chan) | |
dc.contributor.author | Yu-Chiuan Bau | en |
dc.contributor.author | 鮑昱全 | zh_TW |
dc.date.accessioned | 2021-06-16T04:03:38Z | - |
dc.date.available | 2020-03-12 | |
dc.date.copyright | 2015-03-12 | |
dc.date.issued | 2014 | |
dc.date.submitted | 2014-10-06 | |
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A domain insertion in Escherichia coli GyrB adopts a novel fold that plays a critical role in gyrase function. Nucleic Acids Res. 41, 7830-7844 (2010). 30. Marcus Bantscheff, Verena Weiss, and Michael O. Glocker. Identification of linker regions and domain borders of the transcription activator protein NtrC from Escherichia coli by limited proteolysis, in-gel digestion, and mass spectrometry. Biochemistry 38, 11012-11020 (1999). 31. Roland Pfoh, Angela Li, Nilmadhab Chakrabarti, Jian Payandeh, Regis Pomes, and Emil F. Pai. Structural asymmetry in the magnesium channel CorA points to sequential allosteric regulation. Proc. Nati. Acad. Sci. USA 109, 18809-18814 (2012). 32. Ravi Kambampati, Carla Pellegrino, Anthony Paiva, Lee Huang, Liane Mende-Mueller, and Kalpana Chakraburtty. Limited proteolysis of yeast elongation fctor 3. J. Biol. Chem. 275, 16963-16968 (2000). 33. Lionel Costenaro, J. Gunter Grossmann, Christine Ebel, and Anthony Maxwell. 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Esken, Ilaria Giordano, Jennifer L. Hoover, Jianzhong Huang, Graham E. Jones, Senthill K. Kusalakumari Sukmar, Claus Spitzfaden, Roger E. Markwell, Elisabeth A. Minthorn, Steve Rittenhouse, Michael N. Gwynn, Neil D. Pearson. Novel hydroxyl tricyclics (e.g., GSK966587) as potent inhibitors of bacterial type IIA topoisomerases. Bioorg. Med. Chem. Lett. 23, 5437-5441. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/55459 | - |
dc.description.abstract | DNA分子記錄遺傳資訊,但只有DNA卻並不足以執行生物功能。絕大多數牽涉核酸細胞生理活動,諸如DNA複製、轉錄作用、DNA的同源重組,以及其他DNA修補系統等等,都需要一系列的蛋白質參與,其中也包含許多種DNA結合蛋白。目前已知多種調控給定DNA結合蛋白活性的方法。調控蛋白可以直接結合至目標DNA序列並物理性的佔據此空間,又或者引起DNA構型變化以藉此干擾DNA-蛋白質間的交互作用。有些調控蛋白則是作為可化學修飾目標DNA片段的酵素,經由修飾DNA達成調控生理代謝途徑的目的。DNA擬態蛋白則是另一種不同的DNA結合蛋白活性調控策略。一般來說,一種DNA擬態蛋白可以結合至特定的一或幾種目標DNA結合蛋白,並避免這些DNA結合蛋白與DNA發生交互作用,進而影響生理代謝途徑。
MfpA蛋白被認為是一種DNA旋轉酶 (一種細菌的IIA型DNA拓樸異構酶) 抑制物,並可以使分枝桿菌屬的細菌對quinolones抗生素的最小抗藥濃度略微上升4-8倍。結晶學分析顯示MfpA蛋白是一種桿狀的二聚體、並具有帶高度負電荷的表面,其外觀讓MfpA看起來像是一段30鹼基對長的雙股DNA。在5 μM的高純度MfpA存在時,DNA旋轉酶活性會受到抑制。而Biacore實驗則表明MfpA與大腸桿菌DNA旋轉酶間的解離常數位在次微莫耳濃度的範圍。基於上述實驗結果,目前的分子嵌合模型認為MfpA應是作為G-segment DNA的競爭物。MfpA抑制DNA旋轉酶活性、並強化細胞抗藥性的分子機制推測是經由阻斷G-segment結合位置,進而避免具有細胞毒性的quinolones穩定化DNA切割中間產物累積。 在本研究中,我們試圖探討MfpA與DNA旋轉酶的複合體結構。在實驗前期,我們建立了MfpA蛋白、以及一系列DNA旋轉酶次單元與融合蛋白的表現與純化系統。初步的高通量結晶條件篩選實驗並沒有觀察到蛋白質晶體或類似蛋白質晶體的物質。等溫熱卡計滴定分析也有被用於探測MfpA與DNA旋轉酶的交互作用,然而蛋白質沉澱在此方法中非常嚴重,因此等溫熱卡計滴定有可能並不適合此兩種蛋白質。實驗後期,我們建立了一套基於液相層析系統的MfpA-DNA旋轉酶複合體生成分析方法。我們已測試了幾種可能的複合體生成模型,不過目前尚未有正面結果。 | zh_TW |
dc.description.abstract | DNA molecule encodes the genetic information, but DNA on its own is not sufficient to exhibit biological functions. Almost all nucleic acid transactions, such as DNA replication, transcription, homologous recombination, and other DNA repair pathways require the involvement of a series of protein components, including numerous DNA-binding proteins. There are many ways to regulate the activity of a given DNA-binding protein. The regulatory proteins may directly bind to the target DNA sequences and occupy the binding sites or introduce conformational change in DNA to interfere with the DNA-protein interactions. In some cases, the regulatory protein functions as an enzyme, which could chemically modify its target DNA to regulate certain biological pathways. DNA mimicry by protein represents another strategy for regulating the activity of DNA-binding proteins. Each DNA-mimic protein can associate with a particular DNA-binding protein to prevent its interaction with DNA.
MfpA was reported as a DNA gyrase, a bacterial type IIA topoisomerase inhibitor that mildly increase quinolones resistance of Mycobacteria by four to eight-folds. Crystallographic analysis revealed that MfpA protein exists as a rod-shaped dimer with highly negatively charged surface, which makes the protein similar to a stretch of 30 base pairs long double-stranded DNA. The gyrase activity can be inhibited in the presence of 5 μM of purified MfpA. Further Biacore experiments indicated that the dissociation constant is at sub-micromolar range for the interaction between MfpA and Escherichia coli gyrase. A docking model was constructed based on these experiment results, by treating MfpA as G-segment DNA competitor. Thus it was proposed that MfpA inhibits the gyrase activity by blocking the G-segment binding site and prevents the accumulation of cytotoxic quinolone-stabilized cleavage intermediates. We attempted to elucidate the structure of MfpA-gyrase complex in this study. In the early stage, we established the expression and purification systems of MfpA, gyrase subunits, and gyrase fusion proteins. Preliminary high-throughput crystallization screening was used in this work, but so far no crystal or crystal-like substances were observed. Isothermal calorimetry titration analysis of the MfpA-gyrase interaction suffered from heavy protein precipitation and may not be suitable for studying these two proteins. In the latter stage, a chromatography-based MfpA-gyrase complex formation analysis procedure was established. Several different complex formation models had been tested, despite lack of success. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T04:03:38Z (GMT). No. of bitstreams: 1 ntu-103-R01442008-1.pdf: 10764534 bytes, checksum: 8f3eecc5959e2cd98d03d2ea4cd4b356 (MD5) Previous issue date: 2014 | en |
dc.description.tableofcontents | 口試委員會審定書 ii
摘要 iii Abstract iv Table of Contents vi List of figures viii 1. Introduction 1 1-1 DNA structure and topology 1 1-2 The interaction between DNA and DNA-binding proteins 3 1-3 The structure and DNA-binding properties of type IIA topoisomerases 4 1-4 The catalytic mechanism of type IIA topoisomerase and IIA enzyme inhibitor antibiotics 6 1-5 The interactions between DNA-mimic and DNA-binding proteins 8 1-6 The structure and function of MfpA 9 1-7 Specific aims 10 2. Materials and methods 11 2-1 Materials 11 2-1-1 Biomaterials, reagents and consumables 11 2-1-2 Instruments and equipment 13 2-2 Methods 13 2-2-1 Construction of expression vectors 14 2-2-2 MfpA expression and purification 15 2-2-3 DNA gyrase expression and purification 18 2-2-4 MfpA:DNA gyrase complex formation analysis 20 2-2-5 MfpA:DNA gyrase complex crystallization screening 21 2-2-6 Isothermal calorimetry analysis 21 2-2-7 Trypsin limited proteolysis 22 3. Results 23 3-1 MfpA expression and purification 23 3-2 DNA gyrase expression and purification 24 3-2-1 The 145 kDa fusion protein 24 3-2-2 The 107 kDa fusion protein 25 3-2-3 The 110 kDa fusion protein 25 3-2-2 The GyrA 59 kDa fragment 26 3-2-2 The full-length GyrB protein 27 3-3 MfpA:DNA gyrase complex formation analysis 27 3-3-1 The oligomeric 107 kDa fusion protein may interact with MfpA non-specifically 27 3-3-2 DNA gyrase may not interact directly with MfpA in regular chromatographic conditions 28 3-4 MfpA:DNA gyrase complex crystallization screening 29 3-5 Trypsin limited proteolysis 29 3-6 Isothermal calorimetry analysis 29 4. Discussion 31 4-1 Optimization of DNA gyrase purification 31 4-2 Methodology in MfpA:DNA gyrase complex formation analysis 32 4-3 The molecular mechanism of MfpA-mediated inhibition of DNA gyrase activity 33 5. Figures 36 References 73 | |
dc.language.iso | en | |
dc.title | DNA擬態蛋白MfpA與DNA旋轉酶之交互作用分析 | zh_TW |
dc.title | Toward the structural and biophysical characterization of the interaction between DNA gyrase and DNA-mimetic protein MfpA | en |
dc.type | Thesis | |
dc.date.schoolyear | 103-1 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 徐駿森(Chun-Hua Hsu),曾秀如(Shiou-Ru Tzeng) | |
dc.subject.keyword | DNA旋轉?,MfpA,DNA擬態,DNA-蛋白質交互作用,fluoroquinolone, | zh_TW |
dc.subject.keyword | DNA gyrase,MfpA,DNA mimicry,DNA-protein interaction,fluoroquinolone, | en |
dc.relation.page | 77 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2014-10-06 | |
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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