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中文論文名稱 利用13C弛緩動力學來討論不同自結合狀態下的螺旋摺疊:以NPY為模型胜肽
英文論文名稱 Main chain dynamics of peptides with different self-association states in aqueous TFE Using Natural Abundance 13C NMR Relaxation experiments: NPY fragments as the model peptides
校院名稱 淡江大學
系所名稱(中) 化學學系碩士班
系所名稱(英) Department of Chemistry
學年度 98
學期 2
出版年 99
研究生中文姓名 劉士豪
研究生英文姓名 Shih-Hao Liu
電子信箱 697160751@s97.tku.edu.tw
學號 697160751
學位類別 碩士
語文別 中文
口試日期 2010-07-01
論文頁數 65頁
口試委員 指導教授-李長欣
委員-鄧金培
委員-陳怡成
中文關鍵字 神經胜肽  圓二色光譜  核磁共振  自結合  弛緩  動力學  無模型法則 
英文關鍵字 NPY  CD, NMR  self-association  Relaxation  dynamic  Model-Free 
學科別分類 學科別自然科學化學
中文摘要 除了在非常低的濃度下,NPY在不同自結合的狀態之間保持著一些平衡,這個結合狀態過程受到pH值,溫度和溶劑等等來自於分子間作用力的影響,它可能也暗示著當與細胞膜的結合時,單體NPY重要的活性結構摺疊的過程,因此,也表示著在那些不同低聚物之間的平衡和那些在分子之間主要的相互作用。為了更近一步了解在不同自結合狀態下的螺旋摺疊,我們利用hNPY片段[21-31]、[20-36]在285K與310K下的13C弛緩動力學來討論不同自結合狀態下螺旋折疊的變化。當溫度從310K下降到283K時,hNPY[21-31]的實驗分子量會明顯的從1.2增加到單體的4倍,而hNPY[20-36]則是維持著單體的2倍且無太大變化。這個聚合狀態變化可由弛緩實驗導出的整體關聯時間(21-31: 0.53 ns→1.97 ns、20-36: 1.39→3.17)明顯的觀察到。另外,從model-free導出的參數(S2、τe、Rex)更深入的了解內部殘基的運動情況。結構之間的差異,動力學應可清楚指出在結合狀態下構型的變化。藉由合成這兩段短胜肽,我們能成功了解NPY螺旋摺疊及各個殘基的動力學行為。
英文摘要 Except at very low concentrations, NPY is in equilibrium between different states of self-associate. The associate process has a complex dependence on pH, temperature and solvent etc. due to intermolecular forces. It may also imply the fundamental process of monomeric NPY induced folding to an active conformation as binding to cellular membrane. Accordingly, it’s important to elucidate the equilibrium between different oligomeric states and the essential interaction between molecules. For further probing helix folding with different self-association states, we performed natural abundance 13C relaxation dynamics with hNPY fragments (from residue 21 to 31 and 20 to 36) at 285K and 310K. As the temperature decrease from 310 to 285K, the apparent molecular weight of hNPY[21-31] is increased from 1.2 to 4 times of monomeric molecular mass and hNPY[20-36] keep 2 times of monomeric mass in theory. The changes in aggregation state is furthermore evident by overall correlation time (21-31: 0.53ns→1.97ns, 20-36: 1.39ns→3.17ns) derived from relaxation measurements. In addition, parameters (S2, τe, Rex) that exported by model-free could get more insight into internal motions of each residues. The differences between structures, dynamics should clearly point to the direction of the conformational changes during associate process. With this synthesized short peptides we could successfully probe the helix folding and dynamic behavior for each residues.
論文目次 目錄:
目錄: I
圖目錄: III
表目錄: VI
一、前言: 1
研究目的 1
1. NMR與蛋白質動態行為 2
2. 無模型法則(model-free approach) 4
3. NPY簡介 5
二、原理: 8
1. 圓二色旋光光譜儀( Circular Dichroism Spectrometer) 8
多胜肽之二級結構與CD光譜 11
2. 核磁共振(Nuclear Magnetic Resonance, NMR) 13
弛緩(Relaxation) 17
NOE效應(Nuclear Overhauser Effect核歐佛豪瑟效應) 19
3. 二維核磁共振原理(2D NMR) 21
DOSY光譜 (Diffusion Ordered Spectroscopy) 25
4. 無模型法則(Model-Free Approach) 27
函數模型選擇(Model selection) 29
三、材料與方法: 30
1. 圓二色光譜(Circular Dichroism) 30
樣品配製 30
儀器 30
實驗條件 30
計算軟體 30
2. NMR 31
樣品配製 31
儀器 31
實驗條件 31
3. 無模型法則(model-free approach)計算 33
四、實驗結果與討論: 36
1. 圓二色光譜測量 37
2. 擴散實驗-分子間的結合狀態 39
3. 13C弛緩實驗 41
4. 討論 49
5. 動力學參數 56
五、結論: 59
六、參考資料: 61



圖目錄:
圖1-1:左圖為蛋白質骨架結構式,右圖為胜肽鍵主要結構。 ...... 3 
圖1-2:左為NPY在水溶液中單體結構,右為NPY與微胞(DPC micelles)結合的結構疊圖,下方為NPY 36個胺基酸序列。<31> .... 5 
圖1-3:pNPY雙體結構具有平行(parallel)與反平行(anti-parallel)兩種形式。<2> ........................................................................................ 6 
圖1-4:NPY與受質結合過程示意圖。<32> ....................................... 7 
圖2-1:光之電場與磁場示意圖。光是由電場(E)與磁場(M)所組成的電磁波且互成直角。 ........................................................................ 8 
圖2-2:光通過平面極化器(polarized) 後產生單一線性光源。 ..... 9 
圖2-3:左、右旋圓偏極光之合成圖。左圖為通過非旋光性物質;右圖是通過右旋光物質後所產生相角(α)。 ....................................... 9 
圖2-4:線性偏極光通過光學活性物示意圖。<34> .......................... 10 
圖2-5:四種不同胜肽結構的CD吸收圖形。<19> .......................... 12 
圖2-6:原子核在磁場B下所產生的磁矩μ會繞著磁場進動,頻率為ω。 ................................................................................................... 13 
圖2-7:(a)為在B0磁場下,在Z軸上的磁矩,(b)為其能階。 .... 14 
圖2-8:原子核吸收RF pulse能量,產生能階狀態的躍遷。 ....... 15 
圖2-9:整個系統磁矩向量總和M0產生示意圖。 ......................... 16 
圖2-10:M0受電磁脈衝偏轉到XY平面。 .................................... 16 
圖2-11:M0在Z軸與XY平面弛緩的簡易圖。 ............................ 17 
圖2-12:Mz’與T1關係圖。 ............................................................... 18 
圖2-13:Mx’-y’與T2關係式與關係圖。 ........................................... 18 
圖2-14:弛緩過程中,原子核能階的變化示意圖。 ...................... 19 
圖2-15:兩個偶極耦合的自旋I和S的弛緩路徑,W0、W1和W2分別表示零量子、單量子和雙量子的躍遷,W指的是躍遷的機率,W0和W2的弛緩路徑稱為交叉弛緩(cross relaxation)。<15> .......... 19 
圖2-16:W0和W2的交叉弛緩(cross relaxation)能階示意圖。<15> ............................................................................................................... 20 
圖2-17:多維脈衝序列。 .................................................................. 21 
圖2-18:二維核磁共振實驗之脈衝序列可分四個時期。 .............. 21 
圖2-19:t1線性增加實驗。 ............................................................... 23 
圖2-20:經過四個時期後,收集到一連串隨t1值變化的S( t1、t2 )。透過兩次傅利葉轉換,可得S(ω1、ω2)。 ...................................... 24 
圖2-21:DOSY實驗的簡易脈衝序列,自旋回訊波序(spin echo sequence, SE)。 ................................................................................... 25 
圖2-22:局部與總體的物理模型示意圖。<20> ................................ 28 
圖4-1:hNPY[20-36]的CD光譜圖。α螺旋比例隨溫度上升而下降。 ............................................................................................................... 37 
圖4-2:hNPY[21-31]的CD光譜圖。α螺旋比例隨溫度上升而下降。 ............................................................................................................... 38 
圖4-3:上圖為黏度與溫度關係,下圖為分子結合狀態與溫度的關係。●為hNPY[20-36]、▲為hNPY[21-31]。 ............................... 40 
圖4-4:hNPY[20-36]在30% TFE-d3 / 70% H2O的[13C, 1H]-T1光譜。 ....................................................................................................... 41 
圖4-5:hNPY[21-31]在50% TFE-d3 / 50% H2O的[13C, 1H]-T1光譜。 ....................................................................................................... 42 
圖4-6:hNPY[20-36]弛緩實驗的曲線擬合(Curve fitting)。上圖為310 K,下圖285 K。 .......................................................................... 43 
圖4-7:hNPY[20-36],由上到下依序是T1、T2、NOE,◆為285 K、■為310 K。 ........................................................................................ 47 
圖4-8:hNPY[21-31],由上到下依序是T1、T2、NOE,◆為285 K、■為310 K。 ........................................................................................ 48 
圖4-9:各殘基S2的比較圖。上圖為hNPY[20-36],下圖為hNPY[21-31],◆為285 K,■為310 K。 ...................................... 51 



表目錄:
表2-1:CD光譜二級結構之特定吸收波長。 ................................. 11 
表3-1:mfinput變數。 ..................................................................... 34 
表4-1:擴散係數(D)、黏度(η)、分子半徑(rs)、分子間結合狀態、與溫度關係表。 .................................................................................. 39 
表4-2:hNPY [20-36]弛緩實驗資料。 ............................................. 46 
表4-3:hNPY [21-31]弛緩實驗資料。 ............................................. 46 
表4-4:hNPY[20-36]骨架動力學參數表。 ..................................... 57 
表4-5:hNPY[21-31]骨架動力學參數表。 ..................................... 57
 
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