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  • 學位論文

臺灣本土嗜熱菌 Meiothermus taiwanensis Lon 蛋白特異區塊酵素功能與結構之研究

Function-Structure Studies on the isolated Domains of Lon Protease from Meiothermus taiwanensis WR-220

指導教授 : 吳世雄

摘要


Lon蛋白酶是一個單一聚合且多功能的酵素,它被高度的保存在生物種之間。Lon蛋白酶分為兩種亞型,LonA及LonB。它們保有ATPase domain歸屬於AAA+ superfamily之中並利用serine-lysine catalytic dyad 作為酵素的活性位置。LonB不同於LonA其具有N端的區段反而具穿膜區塊使之可鑲嵌在細胞膜上。Lon蛋白酶可調節生物體中新陳代謝的過程,適時降解目標蛋白以維持蛋白質的功能與結構的完整性。本篇論文選擇台灣本土嗜熱菌Meiothermus taiwanensis (WR-220),選殖出兩個Lon蛋白酶MtaLonA1(793 a.a)、MtaLonA2(815 a.a)並建構其特異區塊酵素MtaLonA1N (1-312 a.a)、MtaLonA1A (313-585 a.a)、MtaLonA1α (492-585 a.a)、MtaLonA1C (586-793 a.a)、MtaLonA1NA (1-585 a.a)、MtaLonA1AC (313-793 a.a)、MtaLonA2N (1-320 a.a)、MtaLonA2A (321-601 a.a)、MtaLonA2α (508-601 a.a)、MtaLonA2C (602-815 a.a)、MtaLonA2NA (1-601 a.a)、 MtaLonA2AC (321-815 a.a),透過表現與純化探討其結構與功能的特性,進而比較MtaLonA1及MtaLonA2的差異。 結構方面,利用原二色偏光儀分析呈現,所有蛋白皆以α-螺旋為主要的二級結構並且具有完整的三級結構。進一步利用AUC算得MtaLonA1及MtaLonA2分別形成六聚體及四聚體,於原態膠體電泳實驗中,MtaLonA1N、A1NA、MtaLonA2N、A2NA特異區塊酵素具有多聚體的結構。活性方面,MtaLonA1具有Protease、Peptidase、ATPase、Chaperone-like活性;然而,MtaLonA2具有Protease、ATPase、DNA-binding及較弱的Chaperone-like活性。在MtaLonA2的特異區塊酵素中,只有MtaLonA1N不具有DNA-binding的活性;MtaLonA2α呈現較弱的DNA-binding能力。尤其是MtLonA2AC呈現Protease、Peptidase活性及些許的ATPase活性。所有具有N端的蛋白(MtaLonA1N、A1NA、A2N、A2NA)皆有Chaperone-like活性,藉由此結果推斷出N端的序列對於結構聚合及Chaperone-like活性是必要的。我們並發現MtaHUβ為MtaLonA2特有的目標蛋白。基於這些實驗的結果,我們提出MtaLonA1及MtaLonA2具有不同的功能,更重要的是找出特異性的目標蛋白並結晶出完整長度的Lon蛋白酶,以深入了解MtaLonA1及MtaLonA2在生物體之中所參與的細胞調解。

並列摘要


ATP-dependent Lon protease has been known as one of the most evolutionarily conserved proteins in living organisms, which is a homo-oligomeric multi-domain enzyme. Lon proteases are divided into two subfamilies, LonA and LonB. Each possesses both ATPase domain, belonging to the AAA+ superfamily and a proteolytic domain (P-domain) with a serine-lysine catalytic dyad. The difference between LonA and LonB is that LonA contains a large N-terminal domain, whereas LonB has a transmembrane domain that anchors the protein to the membrane. Lon proteases are well-known to regulate the metabolic processes and involve in protein quality control system. In this study, we analyzed the primary sequence of MtaLonA1 (793 a.a) and MtaLonA2 (815 a.a) from Meiothermus taiwanensis (WR-220) and constructed the truncated-domain MtaLonA1N (1-312 a.a), MtaLonA1A (313-585 a.a), MtaLonA1α (492-585 a.a), MtaLonA1C (586-793 a.a), MtaLonA1NA (1-585 a.a), MtaLonA1AC (313-793 a.a), MtaLonA2N (1-320 a.a), MtaLonA2A (321-601 a.a), MtaLonA2α (508-601 a.a), MtaLonA2C (602-815 a.a), MtaLonA2NA (1-601 a.a) and MtaLonA2AC (321-815 a.a). MtaLonA1, MtaLonA2 and their truncated proteins are overexpressed and purified to examine the functional and structural properties. The structural characteristic presented by circular dichroism revealed that all constructs were composed of α-helical as the major secondary structures and all possessed well-defined three-dimensional structures. For quaternary structure, MtaLonA1 reveals mainly as hexamer and MtaLonA2 might be as tetramer. In native-PAGE, MtaLonA1N-, A1NA- and MtaLonA2N-, A2NA-domain exhibited polymeric structures. Functional studies demonstrated that MtaLonA1 shows the protease, peptidase, ATPase and chaperone-like activities; whereas MtaLonA2 shows the protease, ATPase, DNA-binding activity and weaker chaperone-like activities than that of MtaLonA1. Among the truncated proteins of MtaLonA2, only MtaLonA2N (N-domain) shows no DNA-binding activity, and the MtaLonA2α-domain) reveals weaker DNA-binding capability compared to MtaLonA1α. Particularly, MtaLonA2AC has peptidase activity, protease activity, but only slight ATPase activity. MtaLonA1N-, MtaLonA1NA-, MtaLonA2N- and MtaLonA2NA domain have chaperone-like activities. These results suggested that N-terminal sequence is essential for oligomerization and chaperone-like activities. We found out the substrate, MtaHU β is specific to the MtaLonA2. Based on these results, we propose that MtaLonA1 and MtaLonA2 may have different functions and it is important to find out the specific natural substrates and crystallize structures of full-length enzymes.

參考文獻


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