Please use this identifier to cite or link to this item:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/41676
Title: | 嗜熱短桿菌 Lon 蛋白酶 alpha 區塊與去氧核醣核酸結合模式探討 Elucidation of DNA Recognition Mode for the Lon Protease α-domain from Brevibacillus thermoruber WR-249 |
Authors: | Yu-Da Chen 陳昱達 |
Advisor: | 吳世雄(Shih-Hsiung Wu) |
Co-Advisor: | 徐駿森(Chun-Hua Hsu) |
Keyword: | 嗜熱短桿菌,Lon 蛋白酶,alpha區塊,X-ray晶體繞射,核磁共振, Brevibacillus thermoruber WR-249,Lon,α-domain,X-ray crystallography,NMR, |
Publication Year : | 2011 |
Degree: | 碩士 |
Abstract: | Lon 蛋白酶具有 ATPase、chaperone-like 的活性,屬於一種多功能性的酵素,其主要功能在於降解細胞中錯誤摺疊或是受損的蛋白質,並在原核與真核生物的粒線體中均廣泛的被保留下來。先前的研究中證實了嗜熱短桿菌WR-249的 Lon 蛋白酶具有 DNA 結合的能力,然而 Lon 蛋白酶與 DNA 結合的模式與其在生理上的意義目前仍不清楚。
蛋白質序列比對分析顯示 Lon 蛋白酶的 alpha 區塊並不相似於目前已知的DNA結合蛋白,有可能為呈現一新型態的DNA辨認模式。由於此發現與之前所熟悉的 DNA 結合區塊均不相同,本論文將運用蛋白質結晶學與多維核磁共振光譜學的技術,希望能進一步瞭解 Lon 蛋白酶 alpha 區塊對於 DNA 的結合模式。首先,利用了坐式蒸氣擴散法 (sitting drop vapor diffusion) ,我們成功獲得了 alpha 區塊的蛋白質晶體,並於同步輻射光源下收集到晶體數據。其晶體之空間群 (space group) 為 P23,晶胞參數 (cell parameter) 為 a = b = c = 93.8Å ; α = 90°, β = 90°, γ = 90°。之後利用分子置換法,成功的解析了嗜熱短桿菌 Lon 蛋白酶 alpha 區塊的晶體結構。結構顯示 alpha 區塊由四段 α-螺旋 (α-helix) 與一對平行的β-摺板 (β-strand) 所構成,與其他已知不同 Lon 蛋白酶的 alpha 區塊非常相似。而透過核磁共振光譜中化學位移的擾動實驗結果,我們發現有三個區段對於 DNA 的辨識與結合有較重要的影響 : (1) 第四段的 α-helix ; (2) 以及連結第一段 α-helix 與第一段 β-strand 的環狀結構 (loop) ; (3) 以及連結第三段 α-helix 與第二段 β-strand 的環狀結構。最後,我們以定點突變實驗與核磁共振擾動實驗的結果為基礎,透過了 HADDOCK 軟體模擬出一個可能的蛋白-DNA複合物結構。 Lon protease is a multifunctional enzyme, and it’s functions include the degradation of damaged proteins, ATPase, and chaperone-like activities. It is highly conserved in prokaryotes and eukaryotic organelles. Our previous studies demonstrate the DNA binding ability as a novel function for a thermostable Lon protease from Brevibacillus thermoruber WR-249. However, the physiological role and structure of DNA binding by Lon was still poorly understood. Bioinfomatic analysis reveals that the α-domain of Bt-Lon should be a new member of DNA binding domains with an uncharacterized recognition mode. With the aim of elucidating the DNA recognition mode of Bt-Lon, NMR technique and protein crystallography were employed to determine the structures of free and DNA-bound forms of α-domain. The α-domain was crystallized by the sitting drop, vapor diffusion method. X-ray diffraction data was collected at a synchrotron-radiation source and belonged to space group P23 , with unit cell parameters a = b = c = 93.8 Å , α = 90°, β = 90° , γ = 90° . The Bt-Lon α-domain contains four α helices and two parallel β strands and resembles similar domains found in a variety of ATPases. NMR chemical shift perturbation experiments suggest that there are three major sites responsible for DNA binding : (1) α4 ; (2) the loop between α1 and β1 ; (3) and the loop between α3 and β2. The feasible model of protein-DNA complex was docking with HADDOCK software based on the mutation and NMR perturbation data. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/41676 |
Fulltext Rights: | 有償授權 |
Appears in Collections: | 生化科學研究所 |
Files in This Item:
File | Size | Format | |
---|---|---|---|
ntu-100-1.pdf Restricted Access | 5.85 MB | Adobe PDF |
Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.