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研究生:傅啟淳
研究生(外文):Chi-Chun Fu
論文名稱:雞蛋白溶菌酶準靜態過程摺疊機制之研究
論文名稱(外文):Reversible folding of hen egg white lysozyme by a quasi-static process
指導教授:錢偉鈞錢偉鈞引用關係謝定國
指導教授(外文):Wei-Jyun ChienDean-Kuo Hsieh
學位類別:碩士
校院名稱:朝陽科技大學
系所名稱:應用化學系碩士班
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:2005
畢業學年度:94
語文別:中文
論文頁數:169
中文關鍵詞:螺旋構型摺疊溶菌酶化學位移核磁共振
外文關鍵詞:noechemistry shifthelixfoldinglysozymenuclear magnetic resonancenmr
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本論文利用核磁共振技術,觀察以似一階態轉變模型引導雞蛋白溶菌酶之摺疊過程,並推論其折疊核心。摺疊之過程乃利用尿素和二硫蘇醣醇先使雞蛋白溶菌酶完全變性後,依循似一階態轉變模型引導蛋白質經由過臨界反應路徑抵其自然構型。本文利用化學位移差值、背骨胺基質子與a-質子之NOE訊號以及氫氘交換實驗監測雞蛋白溶菌酶在摺疊過程中不同階段之構型特徵。
結果顯示未摺疊態溶菌酶(U)僅具微弱的構型特徵。減低二硫蘇醣醇濃度誘導蛋白質形成明顯之螺旋構型,再進一步降低尿素濃度時螺旋構型則更為穩定。當溶液中之尿素完全移除後在序列39~60 片段形成板狀結構構型。改變pH 值或降低甘露醇濃度對蛋白質構型都未造成明顯之影響。在各中間狀態環境下,雞蛋白溶菌酶氮端C6~G22 及N27~F34 都呈現穩定的螺旋構型特徵。加上未摺疊態溶菌酶於動態光散射儀實驗結果顯示蛋白質並沒有完全展開,具有微弱構型傾向。據此可推論氮-端C6~G22 及N27~F34兩片段應為雞蛋白溶菌酶摺疊起始位置。
In terms of the first-order-like state transition model, we study the folding process of the egg white lysozyme by the nuclear magnetic resonance technique, and try to find out its folding core. By adjusting the concentrations of urea, DTT and other chemical reagents, the egg white lysozyme, beginning from the denaturated state, goes through an overcritical reaction path to the native conformation step by step. The characteristic of different stages in the folding process is monitored by measuring the chemistry shift indexes, the NOE signals of the backbone amino protons and α-protons, and the hydrogen-deuterium exchange rate.
The result reveals that the structure of the unfolded state of lysozyme is very weak. Bringing down the concentration of DTT will induce the formation of helix conformation, further lowering the concentration of urea will stabilize the helix conformation. While urea is completely removed from the solution, β -sheet structure appears in the segment of lysozyme with sequence number from 39 to 60. However, the effect of the pH value and concentration of mannitol on the protein conformation is not obvious.
According to the NMR spectroscopy, the segments C6~G22 and N2 ~F34 of the egg white lysozyme present stable helix conformation in each middle status from the unfolded state to the native conformation. Together with the result of
the dynamic light scattering experiment, it is inferred that these two segments, C6~G22 and N27~F34, should be the initiating location of the folding of the egg white lysozyme.
第一章蛋白質結構..........................................1
§ 1-1 蛋白質分子各級結構..................................1
1-2-1 α-helix.............................................3
1-2-2 β-strand............................................5
第二章蛋白質分子摺疊模型.................................16
§2 -1 蛋白質動力學之漏斗式的摺疊(THE FOLDING FUNNEL) ....18
§ 2-2框架模型(FRAMEWORK MODEL) 和疏水摺疊模型(HYDROPHOBIC COLLAPSE MODEL)..........................................21
§ 2-3 蛋白質分子摺疊動力學(FOLDING KINETICS)之雙能態模型(TWO-STATE MODEL)......................................24
§2 -4 似一階態轉變模型(THE FIRST-ORDER-LIKE STATE TRANSITION MODEL) .......................................30
第三章核磁共振、溶菌酶研究.............................. 40
§ 3-1 蛋白質分子摺疊與核磁共振技術.......................40
§3-2核磁共振光譜(NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY;NMR)基本原理.............................................43
3-2-1 化學位移(Chemical shift).........................46
3-2-2 二維核磁共振光譜(2D NMR).........................47
3-2-3 COSY(correlation spectroscopy)...................49
3-2-4 TOCSY(total correlation spectroscopy)............49
3-2-5 DQF-COSY(double quantum filtering COSY)..........50
3-2-6 NOESY(nuclear overhauser effect spectroscopy)....50
§ 3-3 雞蛋白溶菌酶(HEN EGG WHITE LYSOZYME)...............52
第四章材料與方法.........................................61
§ 4-1 材料與實驗條件.....................................61
§ 4-2 蛋白質分子在核磁共振光譜的判讀.....................65
4-2-1光譜譜峰的指認......................................65
4-2-2化學位移指標—二級結構的判斷........................66
4-2-3構型的限制..........................................67
§ 4-3 雞蛋白溶菌酶參考值.................................68
第五章實驗結果...........................................75
§ 5-1一維光譜訊號的判定..................................75
§ 5-2 1H NMR二維光譜共振訊號之判定.......................77
§ 5-3 化學位移指標(CHEMICAL SHIFT INDEX;CSI)............81
§ 5-4 NOE連接和氫氘交換(H/D EXCHANGE)....................85
第六章討論..............................................126
第七章結論..............................................135
參考文獻................................................137
附錄一..................................................145
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