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標題: | 嗜熱短桿菌Lon蛋白酵素對DNA結合特性之研究 DNA-binding specificity of the Lon protease α-domain from Brevibacillus thermoruber WR-249 |
作者: | Yu-Ching Lin 林宇慶 |
指導教授: | 吳世雄(Shih-Hsiung Wu) |
關鍵字: | 嗜熱短桿菌,表面電漿共振,恆溫滴定熱分析, Brevibacillus thermoruber WR-249,Lon,α-domain,Surface Plasmon resonance,Isothermal titration calorimetry, |
出版年 : | 2010 |
學位: | 博士 |
摘要: | Lon蛋白為一個多功能的單一聚合型酵素,且高度保留存在於各種生物體中。先前的研究指出Lon可以維持生物體中蛋白質完整的功能與結構,或是適時降解特定或非特定目標蛋白,進而參予調控體內多種新陳代謝活動。本論文以台灣本土所分離出的嗜熱短桿菌(Brevibacillus thermoruber WR-249)之Lon蛋白(Bt-Lon)為研究對象,探討其與DNA結合之間的關係,並同時與革蘭氏陽性標準菌株-枯草桿菌(Bacillus subtilis)及革蘭氏陰性標準菌株-大腸桿菌(Escherichia coli)之Lon蛋白進行比較,利用電泳凝膠遲滯法(GMSA)分析,得知原核細菌的Lon蛋白主要皆是利用其中的α-domain為DNA結合區域。利用DNase I配合基質輔助電射游離脫附飛行時間質譜(MALDI-TOF MS)分析,我們找到一段與Bt-Lon α-domain結合的DNA片段(5’-CTGTTAGCGGGC-3’),我們將之命名為ms1。再利用表面電漿共振(SPR)及恆溫滴定熱分析(ITC),確認ms1與Bt-Lon α-domain之間具有高專一性結合能力。參考Bt-Lon α domain結構的表面正電荷分佈,我們針對其中部份帶正電荷之胺基酸殘基進行定點突變,再進行與ms1 DNA之結合分析,結果顯示當Arginine 518殘基突變為丙胺酸後,結合力會明顯下降26倍,顯示在Bt-Lon α-domain與ms1 DNA之間的結合上,Arginine 518扮演重要的角色。 The multi-functional, homo-oligomeric, ATP-dependent Lon protease is highly conserved in prokaryotes and eukaryotic organelles. Previous studies have shown that Lon activity is essential for protein quality control and regulation of metabolic processes. Here we examined the DNA-binding activity of the Lon protease α-domains from Brevibacillus thermoruber, Bacillus subtilis, and Escherichia coli. Gel mobility shift assays indicated that the α-domain from Br. thermoruber has the highest DNA affinity. MALDI-TOF mass spectrometry showed that this α-domain binds to the nucleotide sequence 5’-CTGTTAGCGGGC-3’ (ms1). Surface plasmon resonance and isothermal titration calorimetry showed that a double-stranded DNA fragment of this sequence binds to the α-domain; double-stranded DNA fragments with 0 and 50% identity to the binding sequence had lower affinities for the α-domain. Five mutants of the α-domain from Br. thermoruber carrying single mutations (R537A, R546A, R553A, K580A and R584A) were constructed and showed only 1.2–2.0-fold lower DNA binding affinity; one mutant, R518A, displayed 26-fold lower affinity. The Bt-Lon R518A mutant also has lower affinity to DNA than wild type. These results revealed that Arg 518 of the Bt-Lon from Br. thermoruber plays a critical role in the DNA-binding activity. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/45265 |
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顯示於系所單位: | 生化科學研究所 |
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