https://scholars.lib.ntu.edu.tw/handle/123456789/143274
Title: | 嗜熱短桿菌Lon蛋白酶功能與結構之研究 Function-Structural Studies on the Lon Protease from Function-Structural Studies on the Lon Protease from Brevibacillus thermoruber WR-249 |
Authors: | 李岳倫 Lee, Alan Yueh-Luen |
Keywords: | DNA-結合蛋白質;熱穩定性;Lon 蛋白酶;chaperone-like activity;DNA-binding protein;thermostability;Lon protease;AAA+ protein | Issue Date: | 2004 | Abstract: | 維持蛋白質完整弁鉬P結構的恆定,在生物體細胞內扮演著重要角色。這維持恆定的角色大多由同時具備chaperone弁鄔M能量依賴性的寡聚體蛋白酶(oligomeric protease) 來扮演。特別是當生物處在逆境時(如高溫),這些蛋白酶更凸顯出其重要性,因為在這些逆境下,錯誤摺疊或受損害的蛋白質會大量增加且堆積,造成蛋白質恆定異常。本論文即從台灣烏來溫泉菌-嗜熱短桿菌(Brevibacillus thermoruber WR-249)中選殖出其中一種寡聚體蛋白酶-Lon蛋白酶的基因(Bt-lon),利用遺傳工程得到大量蛋白質產物後,鑑定其酵素弁鄐弮鶱酋w性,並進一步探討弁鉬P四級結構之間的關係。 Bt-lon可轉譯出分子量約為88 kDa的Lon蛋白酶(Bt-Lon)。Bt-Lon大致可分成三個弁鈰?domain):N端弁鈰洁B中間ATP水解酶弁鈰洏H及C端蛋白酶弁鈰洁A其中ATP水解酶弁鈰狫晱]含了受質偵測識別弁鈰?sensor- and substrate- discrimination domain, SSD domain)。首先經實驗證明,Bt-lon是一個受熱誘導的熱休克蛋白基因。接著,實驗證明Bt-Lon 是一個具有多弁鄋熙J白酶,包括蛋白質的水解、ATP的水解、chaperone-like弁鄍H及與DNA結合之能力。由部分實驗結果顯示,蛋白質的水解與DNA結合能力的最佳溫度均為50℃,此溫度正是這株嗜熱短桿菌生長的最佳溫度。然而,ATP的水解的最佳溫度則為70℃。後續也顯示蛋白質的水解必須在ATP的存在下才能發揮作用,這確認了Bt-Lon的確是一個ATP依賴性(ATP-dependent)的蛋白酶。而且Bt-Lon對受質的專一性也與大腸桿菌Lon不盡相同。值得一提的是,我們首先提出直接證據證明Lon蛋白酶具有chaperone-like弁遄A它可有效地抑制變性蛋白質的不規則聚集,且是與Bt-Lon本身的濃度有關而與ATP存在與否無關。在熱穩定性的探討方面,利用CD光譜進行熱變性實驗可得到Bt-Lon的Tm值為71.5℃。更進一步將Bt-Lon與炫饃黖?Bacillus subtilis) Lon (Bs-Lon)作序列分析的比較,結果顯示Bt-Lon的熱穩定性可能來自蛋白質較堅固,不易受熱變動或者來自有較多的離子鍵或氫建,不過尚待實驗證明。另一方面,也開始對Bt-Lon的四級結構形成的特性作初步的探討。由分析型膠體過濾層析法(analytical gel filtration chromatography)實驗結果顯示,Bt-Lon是一個含六個次單元體的複合體結構。同樣地,從化學連結實驗(chemical crosslinking experiments)也可得到相同結果。同時也以此實驗探討Bt-Lon四級結構形成的過程與性質。結果發現Bt-Lon六元體的形成過程傾向二元體 « 四元體 « 六元體的組合模式。而且也發現維持二元體的主要作用力為疏水性作用力;而整個六元體主要是靠離子交互作用力。 為了進一步了解四級結構與弁鄐孜〞疑鰜Y,我們設計了七個Bt-Lon的刪去突變株蛋白質(truncated mutants),然後利用這些突變株測試蛋白質的水解、ATP的水解、chaperone-like弁鄍H及與DNA結合之能力。同時利用分析型膠體過濾層析法和分析型超高速離心機(analytical ultracentrifugation)來測定這些突變株四級結構的變化。結果顯示Bt-Lon的N端是形成四級結構的必要條件,而且結果也顯示四級結構形成的正確與否也直接影響其弁鄋犒B作。因為去掉N端的Bt-Lon突變株不但無法形成四級結構,而且也失去蛋白質的水解、ATP的水解以及chaperone-like活性的弁遄C從這些結果顯示四級結構的形成對於整個Bt-Lon弁鄋滌鶡瑽篝t著著決定性的關鍵角色。出乎意料之外,結果發現Bt-Lon的N端並不會與DNA結合,進一步尋找後發現,Bt-Lon的SSD弁鈰洃~是與DNA結合有關。最後利用電腦模擬結構對此SSD弁鈰牁s發現的弁鈰筆嗾膋滌Q論。 Protein quality control plays key roles in prokaryotic and eukaryotic cells by depicting cellular surveillance systems of structural and functional integrity of proteins inside a cell. Oligomeric ATP-dependent proteases consisting of the chaperone and the protease machinery carry out such quality control. These energy-dependent proteases are especially important under stress conditions because increasing amounts of misfolding and damaged proteins may accumulate during various stresses. A thermostable Lon protease from Brevibacillus thermoruber WR-249 (Bt-Lon) has been cloned and characterized. The Br. thermoruber Lon gene (Bt-lon) encodes an 88-kDa protein characterized with an N-terminal domain, a central ATPase domain including an SSD (sensor- and substrate- discrimination) domain, and a C-terminal protease domain. The Bt-lon is a heat-inducible gene and may be controlled under a putative Bacillus subtilis sA-dependent promoter but in the absence of CIRCE (controlling inverted repeat of chaperone expression). Bt-lon was expressed in E. coli and its protein product was purified. Bt-Lon is a multi-functional enzyme and its functions include the degradation of proteins, ATPase and chaperone-like activities, and DNA binding. The optimal temperature of ATPase activity for Bt-Lon is at 70oC, while the optimal temperature of peptidase and DNA-binding activities is 50oC. The peptidase activity of Bt-Lon increases substantially in the presence of ATP. Furthermore, the substrate specificity of Bt-Lon was found to be different from that of E. coli Lon by using fluorogenic peptides as substrates. Notably, this Bt-Lon protein shows the chaperone-like activity by preventing aggregation of denatured insulin B chain in a dose-dependent and ATP-independent manner. In the thermal denaturation experiments, Bt-Lon was found to display an indicator of thermostability value, Tm of 71.5 oC. Sequence comparison with a mesophilic Lon protease shows differences in the rigidity, electrostatic interactions or hydrogen bonding of Bt-Lon relevant to thermostability. Additionally, the native Br. thermoruber Lon protease (Bt-Lon) showed a hexameric structure as revealed by analytical gel filtration chromatography, and its nature of oligomerization was investigated. The chemical crosslinking experiments revealed that the oligomerization of Bt-Lon proceeds through a dimer « tetramer « hexamer assembly model. Our results also showed that hydrophobic interactions may play important roles in the dimerization of Bt-Lon and that ionic interactions are mainly responsible for hexamer assembly. To identify the roles of individual domains in the oligomerization process and the functional activities, seven truncated mutants of Bt-Lon were designed, expressed and purified. We examined the Bt-Lon mutants using assays that reflect five different aspects of Bt-Lon activity: ATP-independent oligomerization, ATP-dependent proteolysis, ATPase activity, chaperone-like activity, and DNA-binding activity. Our results show that the N-terminal domain is essential for the oligomerization. The truncation of N-terminal domain resulted in the failure of oligomerization and led to the inactivation of proteolytic, ATPase and chaperone-like activities, suggesting that oligomerization of Bt-Lon is a prerequisite for its catalytic and chaperone-like activities. However, the N-terminal region of Bt-Lon is not involved in the interaction with DNA. We further found that the SSD domain with a previously uncertain function is involved in DNA-binding based on gel mobility shift assays (GMSA). |
URI: | http://ntur.lib.ntu.edu.tw//handle/246246/52803 | Other Identifiers: | en-US |
Appears in Collections: | 生化科學研究所 |
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ntu-93-D88242003-1.pdf | 23.31 kB | Adobe PDF | View/Open |
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