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研究生:張祿高
研究生(外文):Lu-Kao Chang
論文名稱:大腸桿菌ClpY 蛋白形成六元環關鍵胺基酸之研究
論文名稱(外文):The study of the key amino acid on ClpY hexamer formation
指導教授:吳蕙芬
指導教授(外文):Whei-Fen Wu
口試委員:徐駿森陳建德陳昭瑩林晉玄
口試日期:2013-07-12
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:農業化學研究所
學門:農業科學學門
學類:農業化學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
語文別:中文
論文頁數:59
中文關鍵詞:蛋白酶ClpYQ六元環蛋白質交互作用
外文關鍵詞:proteaseClpYQhexamerprotein-protein interaction
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蛋白質是重要的生物分子,細菌中具有蛋白質品管系統(protein quality control network),以維持各類蛋白質之生理活性。其中包含有伴隨蛋白(chaperone) 及蛋白酶(protease),伴隨蛋白協助蛋白質對抗構型變化並修正構型已變之蛋白質,蛋白酶則是分解存在時機不適當之蛋白質,或是已經徹底毀損構型而無法挽救之蛋白質。細菌體內的蛋白質降解任務,主要由一系列不同之ATP 依賴型蛋白酶(ATP-dependent protease) 所執行。
ATP 依賴型蛋白酶具兩種功能區塊解構酶(unfoldase) 及胜肽酶(peptidase),前者具有水解ATP 活性將基質解構並傳送胜肽鏈給後者分解,故屬於AAA+ (ATPase associated with various cellular activities) 家族的一員。革蘭氏陰性菌大腸桿菌中,包含ClpAP、ClpXP、ClpYQ、LonA 及FtsH 等五種ATP 依賴型蛋白酶,因為具備形成多元環之特性,ATP 依賴型蛋白酶在生理狀態下皆呈現桶狀結構。
本研究以ClpYQ 作為探討形成多元環的關鍵因素。前人研究顯示,第408個胺基酸tyrosine 對於ClpY 六元環的形成有關鍵影響,突變蛋白ClpY Y408A 不論在有無ATP 或是ClpQ 的存在下,ClpY 形成六元環的能力都較野生型蛋白為差。檢視ClpY 結晶結構1E94,推測Y408 胺基酸其較大的側基,可與鄰近胺基酸產生較多的凡得瓦爾力,因此我們選擇一系列不帶電的胺基酸,建構了側基由小到大的Y408 點突變蛋白。在胞外以聯結試驗觀察ClpY 蛋白在ATPgS 存在下形成六元環的狀況;另利用ClpYQ 之基質SulA,其作為抑制細胞分裂的特性,透過細菌存活程度,檢定胞內ClpYQ 蛋白酶之活性。結果顯示,胞外與胞內的結果趨勢均隨ClpY Y408 側基大小而改變。側基愈小的ClpY 蛋白在胞外形成六元環的能力愈弱;而ClpY Y408 的側基愈小,對細菌生長的限制愈大。以上顯示ClpY Y408 是ClpY 形成六元環的關鍵胺基酸。

Proteins play important roles in the cell. Protein quality control network maintain the physiology function of proteins in bacteria. There are chaperons and porteases in the network. Chaperons help to against the denature and refold the unnature proteins, proteases degrade the damaged proteins or no suitable ones. Degradation relies on ATP-dependent proteases, like ClpAP, ClpXP, ClpYQ, Lon and FtsH in model organism Escherichia coli. Under physiology condition, all of them form homomultimer and then stack to form the barrel shape.
This study exams the key amino acid in ClpY forming the hexamer. Before, we knew ClpY Y408A mutant strictly effect the hexamer formation. In the crytstal structure 1E94, Y408 is very close to the adjacent amino acid R25; therefore we speculate that the van der Waals'' force between ClpY R25 and Y408 might help ClpY hexamer formation. Here, I present some evidence both in vitro and in vivo. I constructed gradient size point mutant on Y408, according to their side chain size of the amino acid. In vitro, the purified mutant proteins corsslink by glutaraldehyde, running the sodium dodecyl sulfate agarose gel electrophoresis (SDS-AGE) to see the 300 kDa hexamer cylinder or not. ClpY Y408W forms more hexamer than Wt, Y408L, Y408C and R25A/Y408A. In vivo, ClpY Y408 mutants constructed on pBAD24, co-transformed with ClpQ and tagged-SulA, which is a substrate of Lon and ClpYQ and can inhibit cell divide, to AC3112 lon- clpYQ-. The wild type ClpYQ can degrade SulA, then cell grow, otherwise the cell would become lethal. Cell viability reveals the Y408W growth equal to wild type, and order as follow: Y408M, Y408L and Y408V/C/A, which growth equal to vector control. Briefly, I show that the side chain of the amino acid of ClpY Y408 mutants affect the function gradually, once the side chain bigger, the hexamer forms more and the cell can grow better.


壹、 前言 1
一、 ATP 依賴型蛋白酶 1
二、 大腸桿菌中ATP 依賴型蛋白酶 2
三、 ClpYQ 之基質 4
四、 ClpYQ 之結構 7
五、 研究動機 9
貳、 材料與方法 10
一、 實驗材料 10
(一) 菌株與質體 10
(二) 藥品與試劑 11
(三) 器材設備 12
(四) 分析軟體 12
二、 方法 13
(一) 目標基因選殖 13
(二) 蛋白質純化 17
(三) SDS蛋白質膠體電泳 20
(四) 蛋白質聯結測試 (cross-linking analysis) 22
(五) 細菌生理活性分析 23
(六) 細菌液態培養密度測定 24
參、 結果 25
一、 以軟體預測影響六元環形成之成因 25
二、 確認ClpY 及其突變株之六元環形成能力 26
三、 胞內ClpYQ 蛋白酶生理活性測試 27
四、 ClpYQ 蛋白酶與基質對細菌生長之影響 28
五、 ClpYQ 蛋白酶對液態培養的細菌密度之影響 29
肆、 討論 30
伍、 結論 33
陸、 參考文獻 34


陳郁君 (2010) 大腸桿菌熱休克蛋白酶ClpYQ 分解基質之研究 (國立臺灣大學農業化學研究所碩士論文)
黃齡誼 (2011) 大腸桿菌ClpYQ 蛋白酶透過ClpY 對其基質SulA 進行辨識、解構及轉送至蛋白酶活性區之研究 (國立臺灣大學農業化學研究所碩士論文)
胡惠婷 (2012) 大腸桿菌莢膜生合成調節蛋白RcsA 為ClpYQ 蛋白酶的基質之研究 (國立臺灣大學農業化學研究所碩士論文)
謝汎擎 (2012) 大腸桿菌ClpYQ 蛋白酶之ClpY I domain 區域双環構造與孔洞區所扮演角色 (國立臺灣大學農業化學研究所博士論文)

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