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標題: | 極端嗜熱古生菌Thermococcus kodakaraensis Lon蛋白酶功能與結構之研究 Function-Structural Analysis of Lon Protease from hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 |
作者: | Hui-Jou Chou 周慧柔 |
指導教授: | 吳世雄(Shih-Hsiung Wu) |
關鍵字: | Lon 蛋白酶,極端嗜熱古生菌,LonB,伴護,六聚體, Lon protease,Thermococcus kodakaraensis KOD1,LonB,Chaperone,Hexamer, |
出版年 : | 2011 |
學位: | 碩士 |
摘要: | ATP-dependent Lon蛋白酶是維持蛋白質功能與結構之衡定的重要成員之一,普遍分布在各種生物體中。在功能方面,Lon能夠分解細胞內不正常累積的蛋白質以及特定的調節蛋白。Lon蛋白酶可大致分為兩種亞型,LonA和LonB。相較於LonB,LonA已廣泛的被學者們研究。先前的研究已經指出極端嗜熱古生菌(Thermococcus kodakaraensis KOD1)的Lon蛋白酶由N端ATP水解酶功能區及C端蛋白水解酶功能區所組成,在宿主體內是以膜蛋白的形式存在,屬於LonB。在本論文中,藉由刪去TK-Lon的穿膜區域,我們設計了1個TK-Lon的突變株蛋白質(TK-LonΔTM),並探討此突變株蛋白質的功能與結構特性。在大量表現並純化出TK-LonΔTM後,實驗證明此突變株蛋白酶具有ATP水解酶與蛋白水解酶活性。利用電泳凝膠位移測定(EMSA),我們發現TK-LonΔTM具有DNA-binding的活性。在伴護功能活性測定實驗中,我們觀察到不論是在加熱誘導或是利用化學試劑破壞使蛋白質不規則聚集的情況下,TK-LonΔTM都能預防變性蛋白質的不規則聚集。在熱穩定性的研究中,利用示差掃描熱分析儀(DSC)可觀察到TK-LonΔTM的熔點(Tm)為98.9℃,我們認為TK-LonΔTM仍然保留原來的極端熱穩定性。在結構方面,遠紫外光圓二色偏光光譜圖顯示TK-LonΔTM以α螺旋為主要二級結構;近紫外光圓二色偏光光譜圖顯示TK-LonΔTM擁有完整的三級結構。進一步利用凝膠過濾層析法、分析級超高速離心儀以及穿透式電子顯微鏡分析TK-LonΔTM的四級結構,我們的結果都顯示TK-LonΔTM會聚集形成六聚體(hexamer),我們認為此突變株與野生型的結構極為相似。這些實驗結果都可以證明Lon蛋白酶的功能與結構特性不只存在於LonA中,也高度保留在LonB中。 ATP-dependent Lon proteases degrade specific short-lived regulatory proteins and are key components of the protein quality control systems in the cell, which are universally distributed in all kingdoms of life. Lon protease can be divided into two subfamilies, LonA and LonB. LonA is well-studied as compared with LonB. Previous studies have shown that Lon protease from Thermococcus kodakaraensis KOD1 (TK-Lon) which belong to LonB is composed of an N-terminal ATPase domain and a C-terminal protease domain and is a membrane-bound protein in its native host. In this study, we designed a TK-Lon mutant protein (TK-LonΔTM) with a deletion of the membrane-anchoring region and characterized its function and structure. TK-LonΔTM was overexpressed in E.coli and purified from soluble fraction displaying ATPase and proteolytic activity. Electrophoresis mobility shift assay showed that TK-LonΔTM has DNA-binding activity. Chaperone activity assay indicated that TK-LonΔTM can prevent aggregation of denature proteins under thermal stress or chemical stress. The melting temperature of TK-LonΔTM was observed at 98.9 ℃ by differential scanning calorimetry, suggesting the extreme thermostable of TK-LonΔTM. Far-UV CD and near-UV CD measurements revealed that TK-LonΔTM consists of α-helices as the major secondary structure and possesses well-defined three-dimensional structure, respectively. Our gel-filtration chromatography assay, analytical ultracentrifugation and transmission electron microscopy all displayed that TK-LonΔTM assembles into hexameric rings that likely mimic the oligomerization state of the holoenzyme. These findings showed that function and structure of Lon protease are conserved in the LonA and LonB subfamilies. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/38695 |
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顯示於系所單位: | 生化科學研究所 |
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