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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 吳蕙芬(Whi-Fin Wu) | |
dc.contributor.author | Sheng-Shiang Peng | en |
dc.contributor.author | 彭聲翔 | zh_TW |
dc.date.accessioned | 2021-06-14T16:43:03Z | - |
dc.date.available | 2010-08-04 | |
dc.date.copyright | 2008-08-04 | |
dc.date.issued | 2008 | |
dc.date.submitted | 2008-07-31 | |
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S. & McKay, D. B. (2003). Structure and reactivity of an asymmetric complex between HslV and I-domain deleted HslU, a prokaryotic homolog of the eukaryotic proteasome. J Mol Biol 330, 185-195. Lau-Wong, I. C., Locke, T., Ellison, M. J., Raivio, T. L. & Frost, L. S. (2008). Activation of the Cpx regulon destabilizes the F plasmid transfer activator, TraJ, via the HslVU protease in Escherichia coli. Mol Microbiol 67, 516-527. Lee, Y.-Y., Chang, C.-F., Kuo, C.-L., Chen, M.-C., Yu, C. H., Lin, P.-I. & Wu, W. F. (2003). Subunit oligomerization and substrate recognition of the Escherichia coli ClpYQ (HslUV) protease implicated by in vivo protein-protein interactions in the yeast two-hybrid system. J Bacteriol 185, 2393-2401. Levchenko, I., Smith, C. K., Walsh, N. P., Sauer, R. T. & Baker, T. A. (1997). PDZ-like domains mediate binding specificity in the Clp/Hsp100 family of chaperones and protease regulatory subunits. Cell 91, 939-947. Miller, J. H. (1972). 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A gene required for nutritional repression of the Bacillus-subtilis dipeptide permease operon. Mol Microbiol 15, 689-702. Smith, C. K., Baker, T. A. & Sauer, R. T. (1999). Lon and Clp family proteases and chaperones share homologous substrate-recognition domains. Proc Natl Acad Sci U S A 96, 6678-6682. Song, H. K., Hartmann, C., Ramachandran, R., Bochtler, M., Behrendt, R., Moroder, L. & Huber, R. (2000). Mutational studies on HslU and its docking mode with HslV. Proc Natl Acad Sci U S A 97, 14103-14108. Thibault, G., Yudin, J., Wong, P., Tsitrin, V., Sprangers, R., Zhao, R. & Houry, W. A. (2006). Specificity in substrate and cofactor recognition by the N-terminal domain of the chaperone ClpX. Proc Natl Acad Sci U S A 103, 17724-17729. Tomoyasu, T., Gamer, J., Bukau, B. & other authors (1995). Escherichia coli FtsH is a membrane-bound, ATP-dependent protease which degrades the heat-shock transcription factor sigma 32. Embo J 14, 2551-2560. Wang, J., Song, J. J., Franklin, M. C. & other authors (2001). Crystal structures of the HslVU peptidase-ATPase complex reveal an ATP-dependent proteolysis mechanism. Structure 9, 177-184. Wu, W.-F., Zhou, Y. N. & Gottesman, S. (1999). Redundant in vivo proteolytic activities of Escherichia coli Lon and the ClpYQ (HslUV) Protease. J Bacteriol 181, 3681-3687. Yamada-Inagawa, T., Okuno, T., Karata, K., Yamanaka, K. & Ogura, T. (2003). Conserved pore residues in the AAA protease FtsH are important for proteolysis and its coupling to ATP hydrolysis. J Bioll Chem 278, 50182-50187. 林佩宜 (2006). 大腸桿菌ClpQ蛋白分子之間的聚合和其C端的功能. (台灣大學農業化學研究所碩士論文). 施如珊 (2004). 大腸桿菌熱休克蛋白ClpY I domain 之突變蛋白及其專一性基質辨識之研究. (台灣大學農業化學研究所碩士論文). 張道遠 (2004). 分析大腸桿菌ClpYQ 蛋白酶之功能性羧基端. (台灣大學農業化學研究所碩士論文). 郭美雪 (2001). 大腸桿菌中ClpYQ 蛋白酶對RcsA 的調控. (台灣大學農業化學研究所碩士論文). 黃吉心 (2006). 大腸桿菌ClpQ 與ClpY 之交互作用與其蛋白酶之活化. (台灣大學農業化學研究所碩士論文). | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/40231 | - |
dc.description.abstract | ATP依賴蛋白酶普遍存在於細菌、古菌以及真核細胞中,這一類型的蛋白酶主要的功能為調節其他蛋白質在細胞中的量,分解不正常堆積的蛋白質或將折疊錯誤之蛋白質調整回正常構型。ClpYQ/HslUV蛋白酶為ATP依賴蛋白酶其中的ㄧ種,是由ClpY(50 KDa)和ClpQ(19 KDa)構成,各自形成六元環後複合成Y6Q6Q6Y6的ClpYQ複合體。ClpY 扮演辨識基質並藉著消耗能量將基質解開送入ClpQ 的角色,而ClpQ的功能則是分解基質。關於ClpY如何辨識基質以及如何與之作用機制尚未完全明瞭。
本研究接續前人研究,以大腸桿菌表現蛋白質,以西方墨點法測試ClpY突變株分解基質SulA的能力,MMS (methyl methanesulfonate) test 了解大片段缺失突變對基質辨識與分解基質之能力。結果發現ClpY突變株多無法分解基質,但綜合前人研究顯示只有I domain才是最重要的基質辨識區域。I domain 上的Loop 2 (175-209)為專一辨識與結合的位置並協助基質進入孔洞。此外I domain 突變株, I186N、M187I、A188S、E193L/E194L、Q198L/Q200L、L199Q及N205K 對於基質辨識作用有明顯強弱區別,說明loop 2 對基質辨識的重要性。 | zh_TW |
dc.description.abstract | ATP-dependent protease generally exist in Bacteria, Archaea and Eucarya. They plays an essential role in controlling the levels of key regulatory proteins and in the elimination of abnormal polypeptides. ClpYQ(HslUV), an (50 KDa) ATPase ClpY and a (19 KDa) peptidase ClpQ, is one of these proteases. ClpYQ is a cylindrical complex, in which two ClpQ hexamers are in an inner core and a hexameric-ClpY is on both side. It is unclear about the mechanisms of how the ClpY recognizes, binds and translocates the specific substrates to ClpQ and the ClpQY degrades the substrates.
In this study, using Western blot analysis was used to analyze recognition and binding substrate region in ClpY mutants and MMS test to detect the SulA degradation by ClpY deletion mutants. Most ClpY mutants can not degrade substrate. Compared with previous research, only I domain could be the recognition region. Loop 2 (175-209 aa) in I domain is the specific recognition and binding site also helped delivering substrate into pore site. I domain mutants: I186N, M187I, A188S, E193L/E194L, Q198L/Q200L, L199Q and N205K presented distinguished binding affinity with substrate explained how important loop 2 was. | en |
dc.description.provenance | Made available in DSpace on 2021-06-14T16:43:03Z (GMT). No. of bitstreams: 1 ntu-97-R95623006-1.pdf: 3826296 bytes, checksum: 798cda725d6cd424cce779587cbde927 (MD5) Previous issue date: 2008 | en |
dc.description.tableofcontents | 誌謝 i
中文摘要 ii Abstract iii 目錄 iv 表次 vii 圖次 viii 附圖(表)次 ix 第一章、前人研究 1 1.1 蛋白酶簡介 1 1.2 大腸桿菌ClpYQ蛋白酶簡介 2 1.3 雙次元體ClpYQ 3 1.4 大腸桿菌ClpYQ蛋白酶結構分析 4 1.5 ClpYQ基質分析 5 1.6 ClpY與基質的辨識 7 1.7 研究動機與目的 8 第二章、材料與方法 10 2.1研究材料 10 2.1.1 試驗菌株及質體 10 2.1.2 藥品與試劑 10 2.1.3 器材設備 12 2.1.4 分析軟體 12 2.2ㄧ般研究方法 12 2.2.1 質體製備 12 2.2.2 勝任細胞 ( competent cell ) 製備 13 2.2.3 聚合酶鏈鎖反應 (PCR) 13 2.2.4 質體轉殖 15 2.2.5 轉形作用 (transformation) 17 2.2.6 SDS蛋白質膠體電泳 18 2.2.7 西方墨點 (Western blotting) 20 2.3 選殖基因表現系統之建立 23 2.3.1 突變株質體建構 23 2.3.2 載體製備 24 2.3.3 蛋白質誘導表現 25 2.3.4 SulA degradation test: MMS活性測試 25 2.4 前人研究方法 26 2.4.1 酵母菌雙雜交系統 26 2.4.2 β-galactosidase 活性分析 27 第三章、試驗結果 29 3.1 ClpY 基質辨識區域研究 29 3.1.1 ClpY I domain 缺失突變株與融合蛋白 30 3.1.2 ClpY I domain 點突變株 31 3.1.3 ClpY ATP-binding site, pore site 與其他突變株 32 3.1.4 C端突變株 Y408A 32 第四章、討論 33 4.1 融合蛋白YX與C端突變 33 4.2 保守序列ATP-binding site 與 pore site 34 4.3 I domain 的缺失突變 35 4.4 點突變影響親合性 36 4.5 結論: ClpY 的基質辨識區域 37 第五章、參考文獻 38 第六章、表 44 第七章、圖 48 第八章、附圖(表) 54 | |
dc.language.iso | zh-TW | |
dc.title | 大腸桿菌ClpY 對基質SulA專一辨識位之研究 | zh_TW |
dc.title | Study of E. coli ClpY specific substrate recognition sites on SulA | en |
dc.type | Thesis | |
dc.date.schoolyear | 96-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 杜宜殷(Yi-Yin Do),楊啟伸(Chii-Shen Yang),徐駿森(Chun-Hua Hsu),陳佩貞(Pei-Jen Chen) | |
dc.subject.keyword | 大腸桿菌,ATP依賴蛋白脢,ClpYQ,SulA,I domain, | zh_TW |
dc.subject.keyword | E. coli,ATP-dependent protease,ClpYQ,SulA,I domain, | en |
dc.relation.page | 65 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2008-08-01 | |
dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
dc.contributor.author-dept | 農業化學研究所 | zh_TW |
顯示於系所單位: | 農業化學系 |
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