臺灣博碩士論文加值系統

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.

謝誌 i
中文摘要 iii
Abstract iv
目 錄 v
圖目錄 viii
表目錄 x
縮寫表 xi
壹、前言 1
一、Lon 蛋白酶的分類與特徵 1
二、Lon 蛋白酶為一 DNA 結合蛋白 2
三、Lon 蛋白酶中 alpha 區塊 (α-domain) 的結構與功能 2
四、嗜熱短桿菌 WR-249 Lon 蛋白酶相關研究成果 3
五、研究目的 4
貳、材料與方法 5
一、X-ray 晶體繞射技術解析蛋白質結構 5
二、核磁共振技術解析蛋白質結構 5
三、實驗材料 6
1. 蛋白質表現載體之構築 (construct) : 6
2. 菌株及培養系統 : 6
3. 寡核甘酸 (oligonucleotide) : 7
4. 培養基與溶液配方 : 7
5. 蛋白質晶體形成篩選套組 : 7
四、實驗方法 7
1. 轉形作用 (transformation) 7
2. 質體DNA之抽取 7
3. DNA 定序與比對 8
4. SDS 蛋白質膠體電泳分析 (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis, SDS-PAGE) 8
5. 胺基酸序列比對 8
6. 蛋白質小量表現 9
7. 蛋白質大量表現 9
8. 同位素標定蛋白質大量表現 9
9. His-tag 親合純化 10
10. 陽離子交換樹脂純化 10
11. 蛋白質濃度測定 11
12. 蛋白質透析與濃縮 11
13. 蛋白質結晶測試 11
14. 蛋白質結晶條件篩選 12
15. 雙股 DNA 製備 12
16. 蛋白質與 DNA 共結晶條件篩選 12
17. 蛋白質晶體形成條件微調 13
18. 抗凍劑選擇 13
19. X-ray 晶體繞射數據收集及處理 (Data collection and processing) 13
20. 蛋白質結構建立 14
21. NMR 實驗 14
22. NMR資料處理 15
23. 骨架共振頻率判讀 (Backbone resonance assignment) 15
24. 利用 NMR 擾動實驗分析蛋白質與 DNA 作用情形 16
25. 利用分子對接軟體模擬蛋白與 DNA 複合物結構 16
26. DAPI 螢光染劑取代實驗 17
參、實驗結果 18
一、利用 X-ray 晶體繞射技術探討 Bt-Lon α-domain 結構 18
1. 胺基酸序列比對 18
2. 蛋白質表現與純化 18
3. 蛋白質晶體培養 19
4. x-ray繞射數據分析與單位晶格判斷 20
5. Bt-Lon α-domain 蛋白結晶結構的建立 20
6. Bt-Lon α-domain 與 DNA 共結晶晶體培養 21
二、利用 NMR 技術探討 Bt-Lon α-domain 與 DNA 結合模式 22
1. 同位素標定 Bt-Lon α-domain 表現與純化 22
2. Bt-Lon α-domain骨架光譜判讀 23
3. 利用 NMR 擾動實驗分析蛋白質與 DNA 作用情形 24
4. 利用分子對接軟體模擬蛋白與 DNA 複合物結構 24
5. DAPI 螢光染劑取代實驗 26
肆、討論 28
一、利用X-ray晶體繞射技術探討 Bt-Lon α-domain 結構 28
二、利用 NMR 技術探討 Bt-Lon α-domain 與 DNA 結合模式 29
三、未來工作 31
伍、圖表 32
陸、參考文獻 61
柒、附錄 64

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