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研究生:林育如
研究生(外文):Lin, Yu-Ju
論文名稱:Proline衍生物對小螺旋蛋白的穩定性探討
論文名稱(外文):Thermodynamic consequences of incorporating 4-substituted proline derivatives into a small helical protein
指導教授:洪嘉呈洪嘉呈引用關係
指導教授(外文):Horng, Jia-Cherng
學位類別:碩士
校院名稱:國立清華大學
系所名稱:化學系
學門:自然科學學門
學類:化學學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:中文
論文頁數:95
中文關鍵詞:雞絨毛蛋白proline 衍生物
外文關鍵詞:aromatic-proline interaction
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Proline 可以形成 exo 或是 endo 構型,且由於立體電子效應的關係當 4R- 位置有拉電子基取代時偏好 Cγ-exo 構形, 4S- 位置具有拉電子基取代時則偏好 Cγ-endo 構形。雖然 proline 不存在於 α-螺旋結構中,但他們常出現在 α-螺旋結構的氮端,所以我們以具有36個胺基酸的雞絨毛蛋白 ( HP36 ) 作為研究之蛋白模型。 HP36 為一個小的螺旋蛋白由三個 α-螺旋結構所組成且在其碳端的 α-螺旋上有 proline (Pro62) 位於此 α-螺旋結構的氮端。在 HP36 的原始結構中, Pro62 為 Cγ-exo 構形且會與 Trp64 有 aromatic-proline 作用力。本論文將 4 號位置具拉電子基團取代的 proline 衍生物包 (2S,4R)-4-hydroxyproline (Hyp), (2S,4R)-4-fluoroproline (Flp), (2S,4R)-4-methoxyproline (Mop), (2S,4S)-4-hydroxyproline (hyp), (2S,4S)-4-fluoroproline (flp), and (2S,4S)-4-methoxyproline (mop) 置換在雞絨毛蛋白中 Pro62 的位子,觀察 proline 的構型對 aromatic-proline 作用力和螺旋結構穩定性的影響。由 CD 和 NMR 實驗結果顯示被置換過的 HP36 其結構與 HP36 的原始結構 (wild type) 差不多,由 CD 變溫實驗與化學誘導變性實驗得知除了 flp 取代外,其他 proline 衍生物的取代均造成比原始結構不穩定。我們實驗結果顯示,以 proline 的衍生物置換 Pro62 不只有立體電子效應會影響 aromatic-proline 作用力, HP36 穩定性還會受到因 proline 衍生物嵌入所造成之立體障礙疏水性作用力的影響。

中文摘要 I
Abstract II
謝誌 IV
目錄 V
圖目錄 VII
表目錄 IX
一、 前言 1
1-1雞絨毛蛋白 (Villin) 簡介 1
1-2 Headpiece 介紹 5
1-3 立體電子效應 8
(1) 立體電子效應對proline 衍生物的影響 10
(2) 立體電子效應對α-螺旋蛋白穩定度的影響 12
1-4 固相胜肽合成法介紹19 14
1-4.1 酯化反應 (esterification) 16
1-4.2 去保護 (deprotection) 16
1-4.3 活化 (activation) 17
1-4.4 耦合 (coupling) 18
1-4.5 切除 (cleavage) 18
1-5 圓二色光譜儀 (Circular Dichroism)22,23 18
1-6 研究動機 23
二、 實驗步驟 25
2-1儀器型號 25
2-2使用藥品 26
2-3 Proline 衍生物的合成與鑑定 30
2-3.1 合成策略 30
2-3.2 化合物的合成 32
2-4 胜肽的合成、純化與鑑定 37
2-4.1 HP36 系列模擬胜肽 37
2-4.2 實驗步驟 38
2-5 光譜實驗 40
2-5.1 UV光譜判定濃度實驗 40
2-5.2 CD 光譜測量 41
2-5.3 NMR 光譜 43
2-5.4 螢光光譜 43
2-6 光譜數據分析 44
2-6.1 CD 變溫實驗的數據處理 44
2-6.2化學誘導變性 CD 實驗的數據處理 46
三、 結果與討論 49
3-1 HP36 系列胜肽 Far-UV CD 光譜探討 49
3-2 HP36 系列胜肽 NMR 實驗 52
3-2.1 1D 1H-NMR 光譜 52
3-2.2 2D 1H-NMR 光譜 54
3-3 HP36 系列胜肽之螢光光譜 60
3-4 變溫實驗與化學誘導變性 CD 實驗探討 62
3-4.1變溫實驗 62
3-4.2 化學誘導變性 CD 實驗 62
3-5 討論 67
第四章 結論 70
參考文獻 (Reference) 71
附錄 75

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