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姓名 陳振瑞(Chen-Jui Chen) 查詢紙本館藏 畢業系所 化學學系 論文名稱 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
(PartⅠ：The aggregation state and topology of melittin in lipid membranePartⅡ：Interaction between the heptad repeat regions of the ectodomain of gp41 )相關論文 檔案 [Endnote RIS 格式]
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摘要(中) Melittin--摘要
Melittin為一種細胞消溶毒素之胜肽，是由26個胺基酸殘基所構成。它是來自歐洲蜂種Apis mellifera之蜂毒中最主要的成分(∼50﹪)；與細胞膜之各項作用已被廣泛的研究，但對於melittin於細胞膜中的寡聚合狀態(oligmerization)與膜中胜肽之定向則有種種不同的解釋。且因melittin所帶+5 - +6價電荷，可能影響到含電荷不同之人造脂類的研究結果，故我們分別使用電中性之人造脂膜(DMPC)與帶負電之人造脂膜(DMPG)來探討melittin於膜中的寡聚合狀態與胜肽在膜中之定向。
我們使用具長距離螢光焠熄(quench)性質之螢光基rhodamine標定於melittin N-端，以探求其於膜中之集合(aggregation)狀態。另利用cobalt對標定於melittin N-端之螢光基NBD的焠熄現象，與acrylamide
會焠熄掉此胜肽所具有tryptophan殘基之螢光的性質，以推得melittin之N-端位置及胺基酸序列19之位置在不同之人造脂膜中之深度；我們並量測melittin於負價微胞(SDS)中之2D NMR圖譜，並以錳離子(Mn+2)滴定來偵測melittin個別胺基酸殘基於於負價微胞(SDS)中之相對位置。
由實驗結果，得知melittin於帶負電之人造脂膜中以近乎單體狀態存在，且胜肽螺旋主軸約平行脂膜平面，但於電中性之人造脂膜中其為2-4個分子的集合狀態，且胜肽螺旋主軸較負電人造脂膜更為垂直脂膜平面。
HIV摘要
Gp41是為人類免疫性缺乏症候群病毒之醣蛋白外鞘的一部分，隨著另一部分之醣蛋白外鞘gp120與人體免疫T4細胞上之CD4受質、和一些輔助受質發生結合後。其將領導gp120/gp41集合體構型上的轉變，而造成gp41形成一三髮夾結構核心。當gp120分離且gp41未行蛋白質摺合成三髮夾結構核心時之狀態，是為抑制愛滋病毒融合藥物作用之目標：分別與gp41之C-terminal區域、或與其N-terminal範圍之三寡聚合物螺旋coiled-coil核心結構發生結合反應，以阻斷可造成細胞融合之三寡聚合物髮夾結構的形成。因gp120其變異性太大，故非我們所要探討之目標。
從gp41裸露於細胞膜外之胺基酸序列得到一些胜肽和縮氨酸類似物能有效的抑制愛滋病毒的融合，其中的一些胜肽：T20 (aa 638-673)、T649 (aa 628-663)，被發現於某一條件下其具有較低的溶解度且於溶液中成均勻混濁高光散射度的狀態。但當加入包含gp41N-terminal區域(NHR)某ㄧ胺基酸序列時可造成T20、T649混濁度的澄清化，利用此簡單的方式與其混濁度變化的靈敏度可找出其gp41N-terminal區域重要胺基酸序列為何？從一系列選自gp41N-terminal區域且採部份胺基酸序列重疊的方式，發現胺基酸片段LLSGIV是與取自gp41C-terminal區域胜肽抑制藥物重要作用的位置。同時發現於混濁度澄清實驗中具活性之胜肽在水溶液狀態，其具有高含量之α-helix及β-sheet二級結構構型。
另T20、T649與包含Leucine zipper區域之胜肽L43L(aa 545-587)分別作用比較，得胺基酸序列628-638於形成最後三寡聚合物髮夾結構是為一重要之位置。且由其反應後之CD光譜圖，得知T20、T649之二級結構將由無序纏繞轉為α-helix結構。同時利用恆溫滴定熱能變化
(ITC)與生物感應晶片分析(Biacore)量測得：T649較T20對gp41N-terminal區域擁有較強之反應能力。摘要(英) Melittin –Abstract
Melittin, a 26-residue cytolytic peptide, is the major component (∼50﹪)of the venom of the European honey bee Apis mellifera. An array of studies have been undertaken to examine the nature of the interaction between melittin and membrane. A survey of the literature on oligomerization state and orientation of melittin in phospholipid bilayers, yielded a conflicting conclusion. Considering the high net charge of +5 to +6 for melittin, it is possible that the charged of lipid may play a role in the interaction with melittin. Therefore we use zwitterionic (DMPC) and anionic (DMPG) phospholipid vesicles to examine oligmerization state and orientation of melittin in different charge lipids.
The highly distance-sensitive fluorescent probe, rhodamine, is selected to label on the N-terminus of melittin. The aggregation numbers of melittin in phospholipid vesicles can be deduced from rhodamine self-quenching effect. In addition cobalt ion was used to quench NBD, which is labeled on melittin N-terminus, and acryamide was used to quench tryptophan of melittin. The membrane insertion depth of the N-terminal melittin in zwitterionic (DMPC) and anionic (DMPG) lipid are studied by quenching N-terminal labeled NBD fluorescence with the quencher, cobalt ions. The relative depth of tryptophan 19 in DMPC and DMPG lipid are compared with acryamide quenching experiment. Additionally, each individual residue of melittin in the position of membrane-mimic SDS micelles is acquired from 2D-NMR experiments through Mn2+ titration.
We concluded that melittin is monomeric in anionic lipid with helix axis nearly parallel to the membrane plane. In marked contrast, in zwitterionic lipid, the number of melittin is dimmer to tetramer with helix axis perpendicular to the membrane plane.
HIV—Abstract
Gp41 is a part of envelope glycorprotein of the human immunodeficiency virus type 1 (HIV-1). Following binding of gp120 to CD4 and a chemokine receptor, conformational changes occur in the gp120/gp41 complex that leads to trimeric hairpin structure core. The pre-hairpin intermediate of gp41 from HIV is the target for two classes of fusion inhibitors that bind to the C-terminal region or the trimeric coiled-coil of N-terminal helices, thereby preventing formation of the fusogenic trimer of hairpins. The gp120 sequence is highly variable, rendering an ineffective target for fusion inhibitors.
A number of peptides and peptide analogs derived from the membrane proximal region of gp41 ectodomain are found to be effective inhibitors of human immunodeficiency virus type 1 (HIV-1)-mediated fusion events. Two of them, T20 (aa 638-673)、T649(aa 628-663), were found disordered and sparingly soluble in water, but became soluble upon mixing with selected, structured peptides from the amino terminal heptad repeat (NHR) region of gp41 using a simple and sensitive method of reduction in the scattering of T20 suspension. From the results on mapping the locus of interaction with T20 by employing partially overlapping peptides derived from NHR, it was concluded that the LLSGIV segment was a critical docking site for the C-terminal peptide of gp41 in its putative inhibitory action. It was also found that peptides capable of solubilizing T20 dispersion have a high content of α-helix, as well as β-sheet, conformation in aqueous solution.
Comparison the turbidity clearance assay between T20-L43L and T649-L43L interaction, it was found the sequence of aa 628-638 likely played an important role of gp41 N-terminal binding. The secondary structure analysis from CD data of T20 and T649 revealed that transformation of random-coil toα-helix on binding of NHR. And Isotheraml Tirtation Calormetry and Real Time Biomolecular interaction data demonstrated that the binding affinity of T649 to gp41 N-terminal domain is stronger than T20.關鍵字(中) ★ 愛滋病醣蛋白質外鞘
★ 蜂毒
★ 混濁度澄清
★ 螢光
★ 核磁共振關鍵字(英) ★ HIV--gp41
★ Biacore
★ fluorescence
★ melittin
★ NMR論文目次 章節目錄
PartⅠ--中文摘要i
--英文摘要ii
PartⅡ--中文摘要iii
--英文摘要v
章節目錄viii
圖表目錄xii
名詞縮寫及胺基酸序列對照表xvii
PartⅠ：The aggregation state and topology of melittin
in lipid membrane
第一章緒論1
第二章原理簡介
2.1 胜肽合成6
2.2 圓二色極譜儀(Circular Dichroism Spectroscopy) 7
2.3 二維核磁共振光譜
(2D Nuclear Magnetic Resonance Spectroscopy) 11
2.4 二維核磁共振於胜肽結構上的應用13
2.4.1 光譜譜峰的指認14
2.4.2 限制條件的找尋16
2.4.3 化學位移(chemical shift) 18
第三章實驗方法
3.1 胜肽樣品準備25
3.2 螢光物之標定26
3.3 Resin的切除(Cleavage) 27
3.4 HPLC實驗-(純化): 28
3.5 SUV製備: 29
3.6 電泳實驗: 30
3.7 Acrylamide Quenching 30
3.8 Cobalt Quenching 31
3.9 Self-quenching of Rho-melittin 32
3.10 Rho-labeled/unlabeled melittin mixed 33
3.11 CD光譜實驗33
3.12 核磁共振實驗34
第四章實驗結果
4.1 SDS-PAGE電泳36
4.2 Self-quenching of Rho-melittin 36
4.3 Rho-labeled/unlabeled melittin mixed 38
4.4 Acrylamide and Cobalt Quenching 39
4.5 CD光譜實驗40
4.6 核磁共振實驗40
第五章討論53
參考文獻58
PartⅡ：Interaction between the heptad repeat regions of
the ectodomain of gp41
第一章緒論
1.1 愛滋病的歷史61
1.2 HIV的生命週期62
1.3 愛滋病的治療62
1.4 gp41構型之改變及其抑制63
第二章原理簡介
2.1 表面電漿子共振(surface plasmon resonance；SPR)原理71
2.2 Biacore之光學原理72
2.3 Biacore感應圖之介紹73
2.4 Biacore之基本組成74
2.4.1. 感應片表面系統74
2.4.2 SPR光學系統78
2.4.3 液體傳送系統78
第三章實驗方法
3.1 胜肽樣品準備及純化83
3.2 渾濁度澄清分析（Turbidity clearance (TC) assay） 83
3.3 CD光譜實驗85
3.4 生物感應晶片分析
（real time biomolecular interaction analyais） 85
3.5 恆溫滴定熱能量測分析
(Isothermal Titration Calorimetry(ITC)) 87
第四章實驗結果
4.1渾濁度澄清分析( Turbidity clearance (TC) assay) 89
4.2 CD光譜實驗93
4.3 恆溫滴定熱能量測分析
( Isothermal Titration Calorimetry (ITC)) 94
4.4 生物感應晶片分析
( real time biomolecular interaction analysis) 95
第五章討論
5.1 反應構形之轉變115
5.2 重要胺基酸序列反應位置尋找117
參考文獻122
附錄一----- Melittin a1
附錄二----- HIV-gp41 b1參考文獻 Melittin
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(Hsien-Ming Kao、Ding-Kwo Chang)審核日期 2003-7-4 推文 plurk
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