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研究生: 吳金汶
Chin-Wen Wu
論文名稱: 稻米凝集素與醣類複合物之X光晶體結構與功能研究
指導教授: 陳俊榮
口試委員:
學位類別: 碩士
Master
系所名稱: 生命科學暨醫學院 - 生物資訊與結構生物研究所
Institute of Bioinformatics and Structural Biology
論文出版年: 2006
畢業學年度: 94
語文別: 中文
論文頁數: 94
中文關鍵詞: 稻米凝集素
相關次數: 點閱:3下載:0
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  • 植物凝集素(lectin or agglutinins)在生物中有很多不同的作用,例如在植物運輸醣分子及儲藏醣類的過程中扮演了重要角色,由於它對醣具有很強的親和性,可當作儲存蛋白。另外,凝集素也與植物抗病性有關,並具有保護植物免受病原菌感染。我們的研究顯示稻米(Oryza Sativa)的凝集素在低氧的環境下,其RNA的量與在高氧(自然環境下)時比較顯著增加。這些因生物或非生物性的壓力而誘發產生的凝集素,通常會產生在細胞質中,它們可能的功能為和核膜上的醣蛋白結合,使核孔變小,這可能和信號傳遞或基因調控有關。我們已經知道稻米凝集素的晶體模型,本篇論文的研究目的為探討稻米凝集素與其專一性結合之甘露糖晶體結構,並找出其結合區域。
    研究結果顯示稻米凝集素的結構主要由十二個β摺板構成,為一錐形結構。在不同條件下長出的晶體,其在空間中的堆積方式也有單體、二聚體及四聚體的不同。分析表面電荷,發現在活性區域有一陰離子口袋區,共由六個胺基酸形成:Gly14,Leu88,Gly133,Thr134,Leu135,Asp137;而在三維空間結構中,甘露糖與胺基酸交互作用的區域就在此口袋區,其中Gly14、Gly133、Thr134、Leu135、Asp137這五個胺基酸會與甘露糖中的O3、O4、O5 和 O6形成氫鍵。這些氫鍵穩定了複合物的結構。


    壹、 緒論 1 1.1 植物凝集素之介紹 1 1.2 不同物種中與甘露糖具有專一性結合的植物凝集素 4 1.3 因壓力而被誘發產生的植物凝集素 5 1.4 研究目的 7 貳、 實驗方法和材料 8 2.1 實驗流程 8 2.2 高純度稻米凝集素的製備 9 2.2.1 稻米的培養(20) 9 2.2.2 凝集素的表現與純化(20) 10 2.3 晶體培養 14 2.3.1 蛋白質的結晶 14 2.3.2 稻米凝集素晶體的培養(20) 14 2.3.3 稻米凝集素與醣複合物晶體的培養 16 2.4 晶體繞射數據的收集和處理 18 2.4.1繞射數據的收集 18 2.4.2繞射圖的取得與數據處理 20 2.5 結構決定的原理和方法 22 2.5.1 相位問題 22 2.5.2 分子置換法 22 2.6 凝集素結構的精調 24 2.7 凝集素複合物結構的決定與精調 26 2.7.1 凝集素複合物結構的決定 26 2.7.2糖在凝集素複合物中可能的結合位置 29 2.7.3凝集素-半乳糖複合物結構中糖的結合位置的決定 35 2.7.4凝集素-2α-Mannobiose單體複合物之糖的結合位置 37 2.7.5凝集素-2α-Mannobiose四聚體複合物之糖的結合位置 41 參、 實驗結果 45 3.1 凝集素專一性分析 45 3.2 凝集素的結構 47 3.2.1 凝集素結構精調後的結果 47 3.2.2 凝集素的結構 49 3.2.3 稻米凝集素和其他甘露糖專一性凝集素序列比對 54 3.3 凝集素與D-半乳糖複合物的結構 55 3.3.1 凝集素複合物結構精調後的結果 55 3.3.2 凝集素-半乳糖複合物的結構 56 3.3.3 凝集素與凝集素-半乳糖複合物結構的疊印 60 3.4 凝集素與2α-Mannobiose單元體複合物的結構 61 3.4.1 凝集素複合物結構精調後的結果 61 3.4.2 凝集素-2α-Mannobiose單體複合物的結構 62 3.4.3 凝集素與凝集素-2α-Mannobiose單體複合物結構的疊印 66 3.5 凝集素與2α-Mannobiose四聚體複合物的結構 67 3.5.1 凝集素四聚體複合物結構精調後的結果 67 3.5.2 凝集素-2α-Mannobiose四聚體複合物的結構 68 3.5.3 凝集素與凝集素-2α-Mannobiose四聚體複合物結構的疊印 74 3.6 凝集素複合物在空間中的堆疊情形 77 肆、 結果討論 79 伍、 參考文獻 82 附錄一、 老鼠之αA crystallin及αAins crystallin晶體的培養 85 附錄二、 人類之α-L-fucosidase 晶體的培養 88 附錄三、 稻米Zinc Finger 蛋白的純化 90

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