以衰減全反射傅氏紅外線光譜技術
研究親膜胜肽干擾脂質膜的機制

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姓名

劉昱攢(Yu-tsan Liu) 查詢紙本館藏

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化學學系

論文名稱

以衰減全反射傅氏紅外線光譜技術研究親膜胜肽干擾脂質膜的機制
(The ATR-FTIR investigation on lipid-affinity peptide induced membrane perturbation )

相關論文

★ 具生物活性寡胜肽之生化性質研究： HIV抑制物之機制及蜂毒與胞膜之作用	★ HIV融合抑制物之作用機制研究
★ 流感病毒血球凝集素(II)膜外區域之物理化學特性分析

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摘要(中)

蛋白質與脂質膜會發生多樣的交互作用，例如包膜病毒表面融合蛋白誘導的膜融合和蜂毒蛋白對細胞膜的消溶作用。其中，病毒表面的融合胜肽、胞外區與穿膜蛋白在病毒進入宿主細胞的過程中扮演了重要角色，蜂毒在脂質膜上的排列狀態與結構與其消溶的活性亦息息相關。本論文以衰減全反射霍氐紅外線光譜將上述幾種胜肽在脂質膜中的性質作一番探討。我們以化學合成方法將HIV和Influenza的融合胜肽、穿膜蛋白兩種胜肽合成出來，用E.coli表現胞外區蛋白，與脂質層混合後使用衰減全反射霍氏紅外線光譜偵測，我們發現融合蛋白的蛋白質二級結構以β結構為主，穿膜蛋白則以α螺旋為主。融合蛋白以傾斜角度約50度插入多層膜內部，穿膜蛋白則如同穿膜一樣以較為垂直的33度插入膜內，但依膜組成不同亦有不同角度出現；我們也發現脂質膜的分子排列均會隨融合胜肽或穿膜蛋白加入而更擾動，不過特別的是當HIV融合胜肽與穿膜胜肽等比例共存在脂質膜中時，脂質膜分子排列會趨於穩定，但Influenza沒有這種情況；而HIV和Influenza均可以在C=O吸收的地方，發現融合胜肽與穿膜胜肽在脂質膜中共存時造成的明顯去水作用。胞外區蛋白結構以α螺旋為主，以接近70度的角度，幾乎平躺在脂質膜上；以外加一段融合胜肽的胞外區蛋白比較，其對於脂質膜的擾動作用並不明顯。
蜂毒蛋白在負電性的脂質膜中，有比中性脂質膜更多的α螺旋結構，以ATR-FTIR和之前文獻推測，蜂毒蛋白應該是以4聚體頭尾相接的聚集方式，單體與空間Z軸呈50度斜躺的方式陷入脂質膜表面，形成四邊形的脂質膜孔洞。本論文利用ATR-FTIR技術驗證闡明，HIV與Influenza在後融合時期的脂質膜狀態有所不同，並驗證蜂毒在脂質膜上的排列模型，應該是由四聚體頭尾構成的四邊形孔洞，與前人所提的模型有部分差異。

摘要(英)

Protein and lipid interaction is important for biology. For example, fusion protein of enveloped virus would mediate the viral membrane fusion, melittin has the hemolytic effect on cell membrane. Fusion peptide, ectodomain and transmembrane domain of viral fusion protein play pivotal roles in viral entry. Aggregation and the orientation of Melittin in membrane may determine its hemolytic activity. We synthesized HIV and Influenza fusion peptide and transmembrane domain by solid phase synthesis, and HIV ectodomain were expressed by E.coli system. Then ATR-FTIR was used to determine the properties of these lipid-affinity peptide and protein in lipid membrane. We found that fusion peptide has major β-sheet structure and transmembrane domain has major α-helix structure. Fusion peptide has tilt angle respect to membrane normal about 50°. Transmembrane domain has almost vertical angle (33°), but the angle may be different by changing the lipid composition.The lipid molecule will be more perturbation by adding the HIV and Influenza fusion peptide or transmembrane domain individually, however, the HIV fusion peptide and transmembrane domain will make lipid membrane molecule more order when they are together in lipid. We also found fusion peptide and transmembrane domain mixture will make membrane more dehydration than other peptide lonely. Finally, the HIV ectodomain can’t almost make any interaction on membrane comparing other lipid-affinity protein.
Melittin has more α-helix in electro-negative membrane, we thought that melittin should be 4-monomer combined by head to tail attach. The monomer was lying on membrane surface by 50° and formed a quadrangle pore. The new melittin model provided a clue to explain melittin pore-formation mechanism.

關鍵字(中)

★ 衰減全反射傅氏紅外線光譜
★ 穿膜蛋白
★ 蜂毒
★ 膜擾動
★ 次序係數
★ 融合胜肽

關鍵字(英)

★ transmembrane domain
★ order parameter
★ fusion peptide
★ melittin
★ ATR-FTIR

論文目次

中文摘要-------------------------------------------------------------------------i
英文摘要-------------------------------------------------------------------------iii
致謝----------------------------------------------------------------------------iv
章節目錄-------------------------------------------------------------------------v
圖目錄----------------------------------------------------------------------------vii
表目錄----------------------------------------------------------------------------ix
名詞縮寫及胺基酸序列對照表----------------------------------------------x
第一章研究背景
1-1病毒表面蛋白對細胞膜造成的融合作用---------------------1
1-2蜂毒蛋白對細胞膜造成的影響---------------------------------3
1-3 Melittin在脂質膜中的排列--------------------------------------6
1-4研究動機------------------------------------------------------------6
第二章原理簡介
2-1傅氏轉換紅外線FTIR---------------------------------------------10
2-2 ATR-FTIR----------------------------------------------------------14
2-3 Polarized ATR-FTIR與生物分子的傾斜角-------------------15
第三章實驗方法
3-1 Peptide樣品製備---------------------------------------------------33
3-2 蛋白質表現--------------------------------------------------------38
3-3 peptide與蛋白質定量---------------------------------------------39
3-4 SDS-PAGE----------------------------------------------------------40
3-5 SUV製備------------------------------------------------------------40
3-6 ATR-FTIR實驗操作----------------------------------------------41
第四章實驗結果
4-1 1700~1600 cm-1 Amide I區間------------------------------------43
4-2 C=O 1730 cm-1振動區間-----------------------------------------44
4-3 3000~2800 cm-1區間CH2 symmetric stretching---------------45
4-4 Polarized ATR-FTIR 的amide I區間分析---------------------47
4-5 HIV與Influenza的穿膜蛋白SDS-PAGE 分析-------------48
4-6 結果總結-----------------------------------------------------------48
第五章討論------------------------------------------------------------76
參考文獻------------------------------------------------------------------------84

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70. 黃維寧，眼鏡蛇心臟毒素在細胞膜形成孔洞之機制研究，國立清華大學，博士論文，民國92年
71. 陳振瑞，具生物活性寡胜肽之生化性質研究：HIV抑制物之機制及蜂毒與胞膜之作用，國立中央大學，碩士論文，民國92年

指導教授

張定國(Ding-Kwo Chang)

審核日期

2007-7-27

推文