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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 吳蕙芬(Whei-Fen Wu) | |
| dc.contributor.author | Fan-Ching Hsieh | en |
| dc.contributor.author | 謝汎擎 | zh_TW |
| dc.date.accessioned | 2021-05-17T09:14:01Z | - |
| dc.date.available | 2017-08-20 | |
| dc.date.available | 2021-05-17T09:14:01Z | - |
| dc.date.copyright | 2012-08-20 | |
| dc.date.issued | 2012 | |
| dc.date.submitted | 2012-08-17 | |
| dc.identifier.citation | 陸、參考文獻
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/6465 | - |
| dc.description.abstract | 摘要
ATP依賴型蛋白酶對於控制關鍵調控蛋白表現量及降解異常蛋白質以維持微生物體內正常生理活動扮演著重要的角色。ClpYQ蛋白酶為ATP依賴型蛋白酶其中的一種由ClpY與ClpQ二種次單元體所組成。ClpY具有ATPase與chaperone的功能而ClpQ為一胜肽酶。ClpY可以自身形成的六元環而與ClpQ十二元體上下相接形成啞鈴狀的聚合分子。ClpY主要負責辨識、解構與運送基質SulA至ClpQ十二元體的活性中心進行分解。ClpY單元體由N、 I 與C三個domain組成各有其獨特的活性而I domain區域突出於N與C domain之外，其主要的功能尚未有清楚的界定。本研究發現ClpYQ蛋白酶對基質MBP-SulA的辨識結合主要是由loop2結構來進行。而I domain loop1(aa.137-150) 結構則是可能在ClpY在與MBP-SulA辨識結合後，ClpYQ蛋白酶進行降解作用時，阻擋多餘的MBP-SulA與ClpY六元環結合的功能。在pore I/loop2雙缺失突變蛋白ΔP1L2(aa 90-93；aa 175-209)在in vitro pull down實驗中有與MBP-SulA最低的相對活性，顯示基質的辨識結合由pore I與loop2共同負責。另外ClpY可以在沒有ATP存在下與SulA進行辨識結合，而且當ClpY形成六元環後只能與少量的MBP-SulA結合。綜合以上結果可推論出ClpY與SulA進行辨識結合的步驟：即ClpY在沒有ATP存在下與SulA進行辨識結合後，SulA-ClpY在ATP存在下與其他未結合的ClpY形成SulA-ClpY6或SulA-ClpY12的聚合，再與ClpQ十二元體形成SulA-ClpY6Q12聚合體，再進行解構、傳送及降解的步驟。而ClpYY408A突變蛋白無法形成穩定的六元環及ClpYQ聚合體，但仍保有部分的ATPase活性及部分的降解活性，顯示了ClpYQ聚合的穩定度對酵素的活性是重要的。 | zh_TW |
| dc.description.abstract | Abstract
ATP dependent proteases play important roles in controlling the levels of key regulatory protein and in the elimination of abnormal proteins to maintain normal physiological functions of microorgamisms. ClpYQ protease, one of ATP dependent proteases includes two subunits ClpY and ClpQ. ClpY acts as an ATPase and chaperone and ClpQ is a peptidase. ClpY is capble of forming a hexamer ring docked with ClpQ dodecamer to constitute a dumbbel-shaped complex. ClpY is responsible to recognize, unfold and traslocate substractes into the proteolytic site of ClpQ for degradation. Besides, ClpY is divided into three domains N, I and C domain. Each domain has it’s own distinct activity. I domain, a unique protruding domain of ClpY, is unclear for its function. In this study, our results demonstrated that ClpY I domain loop2 is responsible for the initial gripping of SulA and loop1 acts as a lid that is likely to prevent an excess of substracts binding for ClpY when ATP is present. ΔP1L2(aa 90-93；aa 175-209) showed the lowest binding activity with MBP-SulA at in vitro pull-down assay and these results indicated that pore I and loop2 are most responsiable for substrates binding. In addition, ClpY was capble of recognizing MBP-SulA wthout ATP to form SulA-ClpY complex and the ClpY hexamer can only bind a MBP-SulA molecule when ATP is present. These results also indicated that ClpY was capble to recognize SulA wthout ATP and formed SulA-ClpY complex for increasing its local surrounding substrate’s concentration and likely subsequently formed SulA-ClpY6 or SulA-ClpY12 complex docked with ClpQ for degradation when ATP is present. Y408A is not capble to form stable hexamer and ClpYQ oligomer maintains partial ATPase activity and degradation activity indicating that ClpYQ oligomerization is important for the enzyme activity. | en |
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| dc.description.tableofcontents | 目錄
論文審定書……………………………………………………………………………i 謝誌……………………………………………………………………………………ii 摘要………………………………………………………………………………iii Abstract………………………………………………………………………………iv 目錄…………………………………………………………………………v 表目錄………………………………………………………………………………vii 圖目錄……………………………………………………………………viii 附圖目錄…………………………………………………………………………x 壹、 前言……………………………………………………………………………1 一、 能量依賴蛋白酶………………………………………………………………1 二、 ClpYQ蛋白酶…………………………………………………………………2 三、 ClpQ結構及功能……………………………………………………………3 四、 ClpY結構及功能……………………………………………………………4 五、 ClpYQ蛋白酶與ATP之間的交互作用………………………………………6 六、 ClpY與ClpQ的交互作用……………………………………………………7 七、 ClpYQ蛋白酶之基質…………………………………....................9 八、 ClpYQ蛋白酶對於SulA之辨識……………………………………10 九、 研究動機與目的…………………………………………………………11 貳、 材料與方法……………………………………………………………………12 一、實驗材料…………………………………………………………………12 (一)菌株與質體…………………………………………………………………12 (二) 藥品與試劑………………………………………………………………12 二、方法………………………………………………………………………14 (一) 目標基因選殖……………………………………………………………14 (二) 蛋白質純化……………………………………………………………17 (三) SDS蛋白質膠體電泳……………………………………………………20 (四)西方墨點分析……………………………………………………………22 (五) 蛋白質活性測試…………………………………………………………26 (六) ATPase活性測定……………………………………………………………27 (七)等溫滴定量熱法………………………………………………………28 (八) 蛋白質聯結測試…………………………………………………………28 (九) Pull down assay……………………………………………………………29 (十)表面電漿共振生物分子感測法…………………………………………30 参、結果……………………………………………………………………………31 一、ClpY與ClpQ及其突變蛋白純化…………………………………………..31 二、ClpY及其突變蛋白對於ClpYQ分解MBP-SulA活性的影響………………31 三、以 in vitro pulldown探討ClpY對於MBP-SulA辨識結合的區域………34 四、以蛋白質聯結測試觀察ClpY及ClpY與ClpQ的聚合………………………38 五、ClpY T87I ATPase突變蛋白的定性分析…………………………………40 六、C domain突變蛋白ClpY Y408A的定性分析……………………………41 七、以表面電漿共振生物分子感測法進行即時偵測MBP-SulA與ClpY、pore I缺失突變蛋白與雙缺失突變蛋白的親和力……………………………… 43 肆、討論……………………………………………………………………………44 伍、結論……………………………………………………………………………49 陸、參考文獻………………………………………………………………………50 表目錄 表一、本論文所使用的菌株………………………………………………………55 表二、本論文所使用的質體………………………………………………………56 表三、本論文所使用的引子………………………………………………………57 表四、ClpY及其缺失突變蛋白於胞外降解MBP-SulA的相對活性………………58 表五、在不同核甘酸存在下ClpY與MBP-SulA在in vitro pulldown試驗中相對活性……………………………………………………………………………59 表六、以表面電漿共振生物分子感測法(Surface plasmon resonance assays)進行即時偵測MBP-SulA與ClpY及其缺失突變蛋白之間的親和力……………60 表七、以表面電漿共振生物分子感測法(Surface plasmon resonance assays)進行即時偵測於有ATP下MBP-SulA與ClpY及其缺失突變蛋白之間的親和力……………………………………………………………………………61 圖目錄 圖一、突變蛋白及及其突變區域位置………………………………………………62 圖二、帶有His tag ClpY與ClpQ的蛋白質純化…………………………………63 圖三、帶有MBP tag SulA蛋白質的純化…………………………………………64 圖四、純化帶有N端His tag的ClpYΔI domain缺失突變蛋白質………………65 圖五、純化帶有N端His tag的ClpY ΔD-loop缺失突變蛋白質…………………66 圖六、ClpYQ蛋白酶於胞外對MBP-SulA降解實驗………………………………67 圖七、ClpYQ與ClpY-ClpQE61C對MBP-SulA的降解實驗……………………68 圖八、ClpY I domain缺失突變蛋白對MBP-SulA的降解實驗……………………69 圖九、 ClpY ΔD-loop缺失突變蛋白對MBP-SulA的降解實驗及其蛋白質穩度.70 圖十、ClpY I domain loop2點突變蛋白對MBP-SulA的降解實驗…………71 圖十一、以不同核苷酸進行ClpY與MBP-SulA的in vitro pulldown實驗………72 圖十二、以 in vitro pulldown分析ClpQ對ClpY與MBP-SulA的辨識結合的影響…………………………………………………………………………73 圖十三、在有或無ATP下以 in vitro pulldown分析ClpYΔI+7Gly缺失突變蛋白與MBP-SulA辨識結合…………………………………………………74 圖十四、在有或無ATP下以 in vitro pulldown分析ClpYΔL1缺失突變蛋白與MBP-SulA辨識結合……………………………………………………75 圖十五、在有或無ATP下以 in vitro pulldown分析ClpYΔL2缺失突變蛋白與MBP-SulA辨識結合……………………………………………………76 圖十六、在有或無ATP下以 in vitro pulldown分析ClpY ΔD-loop缺失突變蛋白與MBP-SulA辨識結合………………………………………………77 圖十七、在有或無ATP下以 in vitro pulldown分析I domain loop2點突變蛋白與MBP-SulA辨識結合…………………………………………………78 圖十八、在有或無ATP下以 in vitro pulldown分析pore I缺失突變蛋白ΔP1與MBP-SulA辨識結合……………………………………………………79 圖十九、在有或無ATP下以 in vitro pulldown分析雙缺失突變蛋白ΔP1L1與MBP-SulA辨識結合……………………………………………………80 圖二十、在有或無ATP下以 in vitro pulldown分析雙缺失突變蛋白ΔP1L2與MBP-SulA辨識結合……………………………………………………81 圖二十一、ClpYΔL2缺失突變蛋白與雙缺失突變蛋白ClpYΔP1L2 in vitro pulldown之結果…………………………………………………………82 圖二十二、ClpY pore I缺失突變蛋白及pore I/I domain雙缺失突變蛋白對MBP-SulA的降解實驗…………………………………………………83 圖二十三、以蛋白質聯結測試觀察ClpY自身聚合及ClpY與ClpQ的聚合……84 圖二十四、以蛋白質聯結測試觀察ClpY與MBP-SulA的結合…………………85 圖二十五、以蛋白質聯結測試觀察ClpYΔL1缺失突變蛋白自身聚合及ClpYΔL1缺失突變蛋白與ClpQ的聚合…………………………………………86 圖二十六、以蛋白質聯結測試觀察ClpYΔL2缺失突變蛋白自身聚合及ClpYΔL2缺失突變蛋白與ClpQ的聚合…………………………………………87 圖二十七、ClpY T87I ATPase突變蛋白對MBP-SulA的降解實驗………………88 圖二十八、ClpY T87I ATPase突變蛋白ATPase的相對活性……………………89 圖二十九、以等溫滴定量熱法(Isothermal Titration Calorimetry, ITC) 進行ClpY、ClpYT87I與ATPγS結合測定………………………………90 圖三十、以蛋白質聯結測試觀察ClpY T87I ATPase突變蛋白自身聚合及ClpY T87I ATPase突變蛋白與ClpQ的聚合………………………………91 圖三十一、ADP存在下以蛋白質聯結測試觀察ClpY T87I ATPase突變蛋白自身聚合及ClpY T87I ATPase突變蛋白與ClpQ的聚合…………………92 圖三十二、ClpY Y408A突變蛋白對MBP-SulA的降解實驗……………………93 圖三十三、ClpY Y408A突變蛋白ATPase的相對活性……………………………94 圖三十四、以蛋白質聯結測試觀察ClpY Y408A突變蛋白自身聚合及ClpY Y408A突變蛋白與ClpQ的聚合………………………………………………95 圖三十五、在有或無ATP下以 in vitro pulldown分析ClpY Y408A突變蛋白與MBP-SulA辨識結合……………………………………………………96 圖三十六、以表面電漿共振生物分子感測法(Surface plasmon resonance assays)進行即時偵測MBP-SulA與ClpY之間的親和力………………………97 圖三十七、以表面電漿共振生物分子感測法(Surface plasmon resonance assays)進行即時偵測MBP-SulA與ClpYΔP1之間的親和力……………………98 圖三十八、以表面電漿共振生物分子感測法(Surface plasmon resonance assays)進行即時偵測MBP-SulA與ClpYΔP1L1之間的親和力 ………………99 圖三十九、以表面電漿共振生物分子感測法(Surface plasmon resonance assays)進行即時偵測MBP-SulA與ClpYΔP1L2之間的親和力………………100 圖四十、以表面電漿共振生物分子感測法(Surface plasmon resonance assays)在ATP存在下進行即時偵測MBP-SulA與ClpY之間的親和力 ………101 圖四十一、以表面電漿共振生物分子感測法(Surface plasmon resonance assays)在ATP存在下進行即時偵測MBP-SulA與ClpYΔP1之間的親和力…102 圖四十二、以表面電漿共振生物分子感測法(Surface plasmon resonance assays)在ATP存在下進行即時偵測MBP-SulA與ClpYΔP1L1之間的親和力…103 圖四十三、以表面電漿共振生物分子感測法(Surface plasmon resonance assays) 在ATP存在下進行即時偵測MBP-SulA與ClpYΔP1L2之間的親和力…104 圖四十四、ClpY 與MBP-SulA辨識結合流程……………………………………105 附圖目錄 附圖一、大腸桿菌ClpYQ結構圖………………………………………………106 附圖二、大腸桿菌ClpY六元環與單元體結構圖………………………………107 附圖三、大腸桿菌ClpY與ClpQ在無ATP下以凝膠過濾法純化其分子大小之分布………………………………………………………………………108 附圖四、大腸桿菌ClpY pore I site之結構………………………………………109 附圖五、基質促進ClpYQ聚合與降解流程……………………………………110 | |
| dc.language.iso | zh-TW | |
| dc.title | 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 | zh_TW |
| dc.title | The role of ClpY I domain double loops structure and pore site in Escherichia coli ClpYQ protease | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 100-2 | |
| dc.description.degree | 博士 | |
| dc.contributor.oralexamcommittee | 陳昭瑩(Chao-Ying Chen),顏瑞泓(JUI-HUNG YEN),陳建德(CHIEN-TEN CHEN),黃楓婷(Feng-Ting Huang) | |
| dc.subject.keyword | ClpYQ,MBP-SulA,pore I site,I domain,pull down,基質辨識, | zh_TW |
| dc.subject.keyword | ClpYQ,MBP-SulA,pore I site,I domain,pull down,substrate recognition, | en |
| dc.relation.page | 110 | |
| dc.rights.note | 同意授權(全球公開) | |
| dc.date.accepted | 2012-08-17 | |
| 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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