第三章 實驗結果與討論
3.3 Full length BCL2 序列時間軌跡圖（time trace） （Figure A2） . 38
同樣地也對 Full length BCL2 進行 2000 幅的錄影，經由 MatLab 程序分析，發 現此時間軌跡圖可大致分為三類：第一種如同 BCL2MidG4 所觀察到的，為 Cy3 與 Cy5 染料分子的螢光強度劇烈地交互變動，造成 EFRET也產生劇烈的變動，將此 度均有出現，一樣使用 Cross-correlation 分析後可以擬合反相指數衰減曲線，代表 構形有快速動態轉換的現象，而第二種相對慢速的時間軌跡圖，則使用 HaMMy
39
摺疊造成的結果。由此程序還可得知其之間轉換的反應速率以及事件數，如 Table 6。未變化的穩定分子的分類，與 BCL2MidG4 觀察到的現象類似，隨著鹽類濃度 增加，由於狀態趨向穩定因此維持在某狀態的時間軌跡增加，然而與縮短版序列 不同的是，Full length BCL2 所停留的狀態介於 EFRET=0.4 至 0.8 之間，因此造成所 觀察到的 EFRET直方圖分布範圍很廣。
Figure 29. Full length BCL2 相對慢速變化的時間軌跡圖
40
Figure 30. 使用 HaMMy 程式所繪製的機率密度地圖 由上至下分別為 50mM、100mM 及 150mM 鉀離子的濃度
41
[K+]/50mM
Transition (FRET value) Rate (s-1) Number of transitions:
0.4 ⇾ 0.6 11.587 324 0.6 ⇾ 0.4 1.326 324 0.6 ⇾ 0.8 1.122 346 0.8 ⇾ 0.6 0.449 351
[K+]/100mM
Transition (FRET value) Rate (s-1) Number of transitions:
0.4 ⇾ 0.6 10.0009 372 0.6 ⇾ 0.4 1.84762 349 0.6 ⇾ 0.8 1.91507 565 0.8 ⇾ 0.6 0.73936 544
[K+]/150mM
Transition (FRET value) Rate (s-1) Number of transitions:
0.6 ⇾ 0.8 2.68581 282 0.8 ⇾ 0.6 1.12626 281
Table 6. 鉀離子濃度為 50mM、100mM 及 150mM 時，慢速變化軌跡圖使用 HaMMy 軟體 所得到的動力學參數
42 length BCL2 和 BCL2MidG4），縮短版序列在鉀離子為低濃度條件下，所產生的構 形之間有快速變化的情形，隨著鉀離子濃度增加，構形趨向於停留在某一穩定的
43
致力於發展能穩定此構形的小分子藥物，然而這些小分子對於 G-四股結構構形的 穩定可能有選擇性，因此了解到 G-四股結構之間的轉換可有助於這些小分子藥物 進一步的發展。
由於此完整序列可形成的 G-四股結構構形眾多，造成所觀察到的 EFRET分布 太廣，即使使用 HaMMy 軟體仍無法明確的定義其狀態數，因此若要進一部探討 詳細的 G-四股結構構形，尚需要對此序列特定的鹼基對作變性(mutation)，以鑑定 確認各種 G-四股結構的鳥嘌呤的結構。
44
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附錄
Figure A1-1. 鉀離子濃度為 10mM， BCL2 MidG4 EFRET快速變動的時間軌跡圖
52
Figure A1-2. 鉀離子濃度為 50mM， BCL2MidG4 EFRET快速變動的時間軌跡圖
53
Figure A1-3. 鉀離子濃度為 50mM， BCL2MidG4 EFRET快速變動的時間軌跡圖
54
Figure A1-4. 鉀離子濃度為 100mM， BCL2 MidG4 EFRET快速變動的時間軌跡圖
55
Figure A1-5. 鉀離子濃度為 150mM， BCL2MidG4 EFRET穩定狀態的時間軌跡圖
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Figure A1-6. 鉀離子濃度為 200mM， BCL2MidG4 EFRET穩定狀態的時間軌跡圖
57
Figure A2-1. 鉀離子濃度為 10mM，Full length BCL2 EFRET相對慢速變動狀態的時間軌跡 圖
58
Figure A2-2. 鉀離子濃度為 50mM，Full length BCL2 EFRET相對慢速變動狀態的時間軌跡 圖
59
Figure A2-3. 鉀離子濃度為 100mM，Full length BCL2 EFRET相對慢速變動狀態的時間軌跡 圖
60
Figure A2-4. 鉀離子濃度為 100mM，Full length BCL2 EFRET相對慢速變動狀態的時間軌跡 圖
61
Figure A2-5. 鉀離子濃度為 100mM，Full length BCL2 EFRET 穩定狀態的時間軌跡圖
62
Figure A2-6. 鉀離子濃度為 150mM，Full length BCL2 EFRET相對慢速變動狀態的時間軌跡 圖
63
Figure A2-7. 鉀離子濃度為 150mM，Full length BCL2 EFRET穩定狀態的時間軌跡圖
64
Figure A2-8. 鉀離子濃度為 200mM，Full length BCL2 EFRET相對慢速變動狀態的時間軌跡 圖
65
Figure A2-9. 鉀離子濃度為 200mM，Full length BCL2 EFRET穩定狀態的時間軌跡圖
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品名 廠牌 CAS.NO
Acetic acid KANTO 64-19-7
BSA New England BioLabs
Biotin-PEG Laysan Bio,Inc
mPEG-SVA Laysan Bio,Inc
Catalase Sigma-Aldrich 9001-05-2
D(+)-Glucose KANTO 50-99-7
Glucose Oxidase Sigma-Aldrich 9001-37-0
Hydrochloric acid KANTO 7647-01-0
Hydrogen Peroxide 島久藥品 7722-84-1
Neutravidin Thermo
Potassium chloride KANTO 7447-40-7 Potassium hydroxide KANTO 1310-58-3 Potassium persulfate Acros 7727-21-1 Sodium tetraborate decahydrate Sigma-Aldrich 1303-96-4
Sodium chloride KANTO 7647-14-5
Sodium hydrogen carbonate KANTO 144-55-8
Sulfuric acid KANTO 7664-93-9
N-[3-(trimethoxysilyl)propyl]ethylenediamine acros 1760-24-3 Tris(hydroxymethyl)aminomethane acros 77-86-1
Trolox® , 97% acros 53188-07-1
Trimethylamine Sigma-Aldrich 75-50-3 Table A 1. 藥品清單