近年來，為了研究生物分子真實的結構與構型變化，侷限效應逐漸受到重視。奈米尺寸的逆微胞與孔洞材料其共同特性包括熱穩定性高，可調變之尺度廣，容易形成均勻度高且大小一致的侷限空間，使之運用於研究侷限效應更具潛力。本研究利用定位自旋標記－電子自旋技術(SDSL-EPR)觀測Tempol以及生物分子Bcl-2-associated X protein(Bax)、T4溶菌酶(T4L)置入逆微胞與孔洞材料中之分子運動特性，並與ㄧ般溶液做比較，希望能具體描述奈米侷限效應對於生物分子之影響。
　　利用連續波長與脈衝式電子自旋共振光譜(Cw/pulse ESR)進行研究，發現生物分子於逆微胞與孔洞材料中，均會造成分子運動速度減緩，代表兩者的確可產生侷限效果；但以結構分析來說，相較於一般純粹溶液，逆微胞中的Bax、孔洞材料中的T4L皆會產生結構遭受擾動的結果；進一步研究帶有標記之分子，一般純粹溶液中的水分子在低溫下會產生結冰的狀態(分子有明顯聚集情況)，而在逆微胞與孔洞材料中於低溫下會維持無定型狀態(glassy amorphous state)。另一方面，加入海藻糖(trehalose)於逆微胞水相中希望能使水相擁擠並加強侷限效應，實驗結果發現加強效果不甚明顯；文獻中曾提到，trehalose會聚集在逆微胞極性部分的位置，因此利用自旋標記脂質研究trehalose與逆微胞壁上的作用力，發現使用Tempo PC以及5-PC均無法得到與文獻相同的資訊，因此驗證文獻之結果需待進一步的深入探究。
　　本研究發現，逆微胞與孔洞材料對於研究侷限效應不全是正面的效果，逆微胞與蛋白質之間的靜電吸引力、蛋白質的選擇(等電位點之考慮)、孔洞材料大小與蛋白質大小關係可能是使用逆微胞與孔洞材料研究侷限效應之額外考量，而這些推測急待後續研究來證實。

Confinement has been demonstrated useful in accelerating the folding process, because of a compact folded protein occupying less volume than an unfolded protein. The scientists have researched on confinement for protein folding, protein diffusion, protein-protein interaction in the recent years. Reverse micelles and mesoporous materials have the common advantages such as high stability, easily tunable size, homogeneous pore structures, and consequently are used to encapsulate molecules for confinement study. In this study, we have explored the confinement effect on biomolecular structure and dynamics by site directed spin label(SDSL) and Cw/pulsed ESR techniques. The molecules we used are tempol, and proteins including Bcl-2-associated X protein(Bax) and T4 lysozyme(T4L).
In Part I, we trap the biomolecules in reverse micelles and mesoporous materials to understand the confinement effect. The motions of biomolecules in nanoconfiment are slower than in bulk solution. The results show the confinement effect exists but the structures of biomolecules are distorted slightly. Moreover, we find that the water molecules in the nanochannels stay on amorphous state but not freezing at cryogenic temperature.
In Part II, we add trehalose in the water pool of reverse micelles to enhance the confinement effect. The spectra change hardly whether the trehalose is added or not. According to the literature, the trehalose interacts with polar surfactant headgroups. We dope the reverse micelles with spin-labeled lipid(tempo PC, 5-PC) to compose reverse micelles. However, the spectra of the spin-labeled lipid change little with trehalose.
Nanoconfinement effects play an important role in protein conformational structure. In this study, we show the conformations of the T4L proteins are somewhat distorted in the mesoporous materials with the pore sizes approximately 8 nm, which is apparently large enough to accommodate the studied proteins. This result is opposed to what we have found for the nano-confined structure of the 26-mer-long polypeptide whose structure was demonstrated to remain unchanged between the bulk solvent and the mesoporous materials. At the current stage, our results point out that the ratio of the pore size and the studied molecular size might be a key to the success for confining a molecule in the nanochannels while leaving its conformation intact. Moreover, it is also possible that the nanoconfinement effects could result in a change of the state in the conformational potential of a protein. Further investigations of the nanoconfinement effect are warranted.

摘要.................................................... I
Abstract................................................III
目錄.................................................... V
圖目錄...................................................VIII
表目錄.................................................. XII
第一章 緒論.............................................. 1
1.1 電子自旋共振(ESR)光譜在生物物理上之應用.................. 1
1.2 侷限效應對生物分子之重要性.............................. 3
1.3 侷限效應(confinement effect)之簡介.................... 5
1.3.1 逆微胞(reverse micelle)之簡介.......................6
1.3.2 奈米孔洞材料(mesoporous material)之簡介..............8
1.4 蛋白質簡介........................................... 10
1.4.1 Bcl-2-associated X protein(Bax)...................10
1.4.2 T4溶菌酶(T4 Lysozyme, T4L).........................12
1.5 研究動機與目的 ........................................13
第二章 儀器介紹與原理...................................... 14
2.1 動態散射式粒徑分析儀(Dynamic Light Scattering, DLS).... 14
2.2 圓二色旋光光譜儀(Circular Dichroism, CD).............. 16
2.3 電子自旋共振光譜儀(Electron Spin Resonance, ESR)...... 21
2.3.1 量測原理........................................... 21
2.3.2 光譜簡介........................................... 24
2.3.3 自旋共振原理....................................... 26
2.3.4 超微細偶合作用(Hyperfine interaction)............... 28
2.3.5 定位自旋標記之應用(application of SDSL-ESR)......... 31
2.3.6 硝基氧自旋標記物的轉動動力學與ESR光譜線形關係........... 34
2.3.7 ESR光譜距離量測.................................... 40
2.3.8 雙重電子-電子共振(DEER)光譜之距離量測................. 41
第三章　樣品製備與儀器測量方法.............................. 46
3.1 實驗流程............................................. 46
3.1.1 Bcl-2-associated X protein (Bax)蛋白的選殖、表現與純化...................................................... 46
3.1.2 T4溶菌酶(T4 Lysozyme, T4L)蛋白選殖、表現與純化 ........49
3.1.3 SDS-PAGE原理與製備................................. 51
3.1.4 Bradford protein–binding assay判定濃度實驗......... 55
3.1.5 定位自旋標記樣品之製備............................... 57
3.1.6 逆微胞溶液包覆生物分子製備............................ 58
3.1.7 利用動態散射式粒徑分析儀(DLS)測量逆微胞之粒徑大小........ 61
3.1.8 ESR樣品製備 ........................................63
3.2 儀器測量方法......................................... 64
3.2.1 動態散射粒徑分析儀(Dynamic Light Scattering, DLS)測量 64
3.2.2 圓二色旋光光譜儀(Circular Dichroism, CD)測量......... 64
3.2.3 連續波型電子自旋共振光譜測量(Cw-ESR).................. 64
3.2.4 脈衝型電子自旋共振光譜測量(pulse-ESR)................. 65
3.3 藥品與儀器........................................... 68
3.3.1 實驗藥品........................................... 68
3.3.2 實驗儀器........................................... 69
第四章　結果與討論........................................ 71
4.1 利用生物分子研究逆微胞與孔洞材料之侷限效應................ 71
4.1.1 Tempol/DI H2O在水溶液與逆微胞中 (AOT/pentane w0=34) 於200 K之Cw-ESR光譜分析.................................... 71
4.1.2 Tempol/DI H2O在逆微胞中 (AOT/pentane w0=34) 於不同溫度之Cw-ESR光譜分析........................................... 73
4.1.3 Tempol在逆微胞中 (AOT/isooctane w0=20) 於不同環境下且經-80℃冷凍前後於不同溫度之Cw-ESR光譜分析...................... 75
4.1.4 Tempol在水溶液與逆微胞中 (AOT/isooctane w0=20) 於200 K之Cw-ESR光譜分析........................................... 79
4.1.5 Bax 67R1在緩衝溶液中於不同溫度之Cw-ESR光譜分析......... 80
4.1.6 Bax 67R1在逆微胞中 (AOT/isooctane w0=20) 於不同溫度之Cw-ESR光譜分析.............................................. 82
4.1.7 Bax 67C於298 K之CD光譜分析......................... 83
4.1.8 Bax 67R1在30% (v/v) glycerol vs. 中孔洞材料SBA15於不同溫度之Cw-ESR光譜分析....................................... 84
4.1.8 不同位點之Bax在40% (v/v) glycerol, 40% (w/w) ficoll vs. 孔洞材料SBA15於不同溫度之Cw-ESR光譜分析..................... 87
4.1.9 利用T4 溶菌酶研究在孔洞材料中的結構變化................ 88
4.1.10 本節討論.......................................... 93
4.2 利用海藻糖 (trehalose) 加強逆微胞之侷限效應.............. 94
4.2.1 Tempol/DI H2O vs. 加入30%(w/v) trehalose在逆微胞中 (AOT/isooctane w0=20) 於不同溫度之Cw-ESR光譜分析........... 95
4.2.2 Tempol/PBS vs. 加入30% (w/v) trehalose在逆微胞中(AOT/isooctane w0=20) 於不同溫度之Cw-ESR光譜分析........... 97
4.2.3 Tempol/30% (w/v) trehalose在逆微胞中(AOT/isooctane w0=20) 經-80℃冷凍後於不同溫度之Cw-ESR光譜分析.............. 98
4.2.4 Bax 67R1 vs. 加入30% (w/v) trehalose在逆微胞中(AOT/isooctane w0=20) 中於不同溫度之Cw-ESR光譜分析......... 101
4.2.5 Bax 67R1 vs 加入30% (w/v) trehalose在逆微胞中(AOT/isooctane w0=20) 經-80℃冷凍後於不同溫度之Cw-ESR光譜分析................................................. 103
4.2.6 本節討論.......................................... 105
4.3 利用spin-labeled lipid研究海藻糖在逆微胞中存在的位置..... 105
4.3.1 Tempo PC/DI H2O vs. 加入30% (w/v) trehalose在逆微胞中(AOT/isooctane w0=20, 10, 5) 於不同溫度之Cw-ESR光譜分析(Tempo PC/AOT=0.0185mol%)..................................... 106
4.3.2 Tempo PC/DI H2O vs. 加入30% (w/v) trehalose在逆微胞中(AOT/isooctane w0=5) 於不同溫度之Cw-ESR光譜分析 (Tempo PC/AOT=0.37 mol%)...................................... 111
4.3.3 Tempo PC/DI H2O vs. 加入30%(w/v) trehalose vs 加入60% (w/v) trehalose在逆微胞中 (AOT/isooctane w0=3)於不同溫度之Cw-ESR光譜分析 (Tempo PC/AOT=0.74 mol%)..................... 114
4.3.4 5-PC/DI H2O vs. 加入30% (w/v) trehalose在逆微胞中(AOT/isooctane w0=5) 於不同溫度之Cw-ESR光譜分析 (5-PC/AOT=0.37 mol%).................................................. 117
4.3.5 本節結論........................................... 119
第五章 結論.............................................. 121
參考文獻................................................. 124

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