臺灣博碩士論文加值系統

人類轉譯後修飾蛋白質SUMO因為其三度空間結構和Ubiquitin相似，故命名為Small Ubiquitin-like MOdifier，目前已經被發現有三種，分別命名為SUMO-1/2/3。本研究主要是以大腸桿菌表現系統，表現重組蛋白質Δ1-8, 94-95 SUMO-2(其N端接10 個Histidine)，分別在4℃和室溫下進行Δ1-8,94-95 SUMO-2的純化及養晶。進一步利用電腦模擬與X-光晶體繞射技術分析其表面性質及三度空間立體結構。純化Δ1-8,94-95SUMO-2是以固定化金屬離子親合性色層分析(IMAC)法分兩個階段進行，濃縮後蛋白質濃度為60 mg/ml。在蛋白質表現量方面，6升的培養液約得到120 mg的蛋白質。再將高濃度和純度的Δ1-8,94-95 SUMO-2以懸滴靜置氣相擴散法進行蛋白質結晶，形成多種外觀不同的晶體。三角形多面體的蛋白質單晶的大小為0.25 x 0.25 x 0.05 mm3，長柱形多面體蛋白質單晶的大小為0.35 x 0.15 x 0.1 mm3。完整的X-光晶體繞射圖譜經初步分析，解析度分別為1.6 Ǻ和1.2 Ǻ。三角形多面體的Δ1-8, 94-95 SUMO-2蛋白質單晶屬於R3 space group，其Unit cell的三邊長為與夾角分別a=b=75.2 Ǻ c=29.2 Ǻ α=β=90° γ=120°。長柱形多面體蛋白質單晶其unit cell的三邊長為a=b=74.9 Ǻ c=33.2 Ǻ，三個夾角則相同。將解析度1.6 Ǻ這組數據做結構測定和精調，結果R-factor＝0.167，R free＝0.188，因此SUMO-2的結構相位精準度相當高。結構比較方面，酵母菌SMT3與SUMO-1 NMR結構Ca原子座標的平均差異值(r.m.s.d.)為2.736 Ǻ，SMT3和SUMO-2晶體結構Ca 原子座標的平均差異值(r.m.s.d.)為1.054 Ǻ，因此SUMO-2和SMT3晶體結構比SUMO-1 NMR結構和SMT3晶體結構更為相似。

The SUMO protein was named Small Ubiquitin-like MOdifier because its 3-D structure was similar to Ubiquitin. In human, three SUMO proteins were discovered, namely, SUMO-1/2/3. The recombinant △1-8, 94-95 SUMO-2 protein with 10 histidine residues at its N-terminus was expressed using E. coli. BL-21(DE3), purified at 4 oC and crystallized at room temperature. The surface properties of human SUMO-1/2/3 proteins and 3-D structure of △1-8, 94-95 SUMO-2 protein were analyzed using computer modeling and X-ray diffraction technology respectively.
The two-step purification by immobilized metal ion affinity chromatography(IMAC) was developed to yield △1-8, 94-95 SUMO-2 protein that reached 60 mg/ml for crystallization. On protein expression, 120 mg protein was obtained from 6 L bacterial growth broth. Crystals of △1-8, 94-95 SUMO-2 were obtained by the hanging-drop vapor diffusion method and many different crystal forms were observed. One of single crystal with triangular plate polyhedron form diffracted to 1.6 Å resolution, the other one with rectangular polyhedron form diffracted to 1.2 Å. Analysis of the diffraction pattern suggests the crystals belong to R3 space group, the former one owned unit cell parameters a= b=75.3 Å, c=29.2 Å, α=90°, β=90°,γ=120°, and the later one owned unit cell parameters a= b=74.9 Å, c=33.2 Å and the same angles respectively.
The R factor and Rfree of refinement are 0.133 and 0.190 with highly precise phase on 3-D structure of SUMO-2 protein. Comparison of crystal structure between human SUMO-2 and yeast SMT3 showed that the r.m.s. deviation of Cα coordinate is 1.054 Å. In addition, comparison of SUMO-1 NMR structure and SMT3 crystal structure showed that the r.m.s. deviation of Cα coordinates is 2.736 Å. Hence, the structures of SUMO-2 and SMT3 are more similar each other than those of SUMO-1and SMT3.

中文摘要 i
英文摘要 ii
第一章 緒論 1
前言 1
壹﹒ SUMO的簡介 2
貳﹒ SUMO基因在不同物種演化上的相關性 3
參﹒ SUMO-1與Ubiquitin蛋白質的相異性 4
肆﹒ SUMO和Ubiquitin作用機轉的相關性 5
伍﹒ SUMO的已知功能和重要性 7
陸﹒ SUMO-1與Ubiquitin蛋白質結構的相似性 11
柒﹒ SMT3-Ulp1的晶體結構 11
捌﹒ 研究目的與策略 12
第二章 人類truncated SUMO-2蛋白質的純化 13
壹﹒ 材料及方法 13
(一)質體的建構 13
(二)利用BL21(DE3)-pET系統誘發truncated SUMO-2蛋白質表現和純化
19
貳﹒ 結果與討論 22
(一)Truncated SUMO-2的質體建構 22
(二)以IMAC純化truncated SUMO-2蛋白質 23
第三章 人類truncated SUMO-2蛋白質的結晶和結構測定 25
壹﹒ 蛋白質的結晶 25
貳﹒ 結構測定 27
參﹒ 結果與討論 29
第四章 人類SUMO蛋白質的表面性質 34
壹﹒ 研究方法 34
貳﹒ 結果與討論 36
結論 39
參考文獻 40
圖表目錄
圖一、Truncated SUMO-2（△1-8, 94-95 SUMO-2）構築於質體pET16b 45
圖二、以UV280 nm分析Ni-NTA純化過程中蛋白質的分佈 46
圖三、以12% Tris-Bis Gel/running buffer(MOPS)初步鑑定蛋白質分子量 46
圖四、質譜分析truncated SUMO-2（△1-8, 94-95SUMO-2）蛋白質分子量 47
圖五、質譜分析truncated SUMO-2（△1-8, 94-95SUMO-2）蛋白質分子量 48
圖六、Truncated SUMO-2養晶的條件 49
圖七、Truncated SUMO-2養晶的條件 49
圖八、Truncated SUMO-2在不同pH Screen條件下形成的晶體 50
圖九、Truncated SUMO-2晶體的X-ray繞射圖譜 51
圖十、Truncated SUMO-2結構的原子模型 52
圖十一、利用程式Grasp建立truncated SUMO-2蛋白質的表面區域 53
圖十二、利用程式Grasp建立truncated SUMO-2蛋白質的結構 53
圖十三、Truncated SUMO-2晶體的晶體的電子密度圖 54
圖十四、SUMO-1 NMR結構、SUMO-2及SMT3晶體結構 55
圖十五、人類SUMO-2/3/1與Ubiquitin的氨基酸序 56
圖十六、Truncated SUMO-1蛋白質分子模型的表面性質 57
圖十七、Truncated SUMO-2蛋白質結構的表面性質 58
圖十八、Truncated SUMO-3蛋白質結構的表面性質 59
圖十九、Truncated SUMO-2蛋白質的二級結構 60
圖二十、Truncated SUMO-1蛋白質分子模型的表面氨基酸組成 61
圖二十一、Truncated SUMO-2蛋白質結構的表面氨基酸組成 62
圖二十二、Truncated SUMO-3蛋白質分子模型的表面氨基酸組成 63
圖二十三、Truncated SUMO-2與Ubuquitin蛋白質的表面性質 64
圖二十四、Truncated SUMO-2與Ubuquitin蛋白質的表面性質 65
表一、Truncated SUMO-2晶體(三角形多面體)X-ray繞射圖譜的初步分析 66
表二、Truncated SUMO-2晶體(長柱形多面體)X-ray繞射圖譜的初步分析 67
表三、Truncated SUMO-2晶體的X-ray繞射實驗數據統計 68
表四、Truncated SUMO-2晶體結構的微調 69
表五、比較SMT3、SUMO-2晶體結構和SUMO-1 NMR結構差異 70
表六、比較SUMO-1與SUMO-2在二級結構氨基酸組成 71
附錄
附圖一、不同物種間SUMO-1/2/3氨基酸序列的相同性和相似性 72
附圖二、SUMO-1與Ubiquitin立體俯視圖 73
附圖三、說明SUMOylation包含的過程及參與反應的酵素 74
附圖四、比較Ubiquitination與SUMOylation作用機轉的差異 75
附圖五、SUMO參與訊號傳遞的功能 76
附圖六、SUMO-1以NMR測定得到10種結構 77
附圖七、pET16b質體 78
附圖八、Truncated SUMO-2的核酸與氨基酸序列 79
附表一、不同物種間SUMO-1/2/3氨基酸序列的相同性和相似性 80
附表二、不同目標蛋白的一般序列（consensus sequence）YKXE 81
附表三、酵母菌SMT3與Ulp1的作用位置和作用力 82

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