環化脂質奈米碟( circularized lipid nanodisc )搭配負電脂質( dimyristoyl phosphatedyl-glycerol, DMPG )提供近似細菌視紫質( bacteriorhodopsin, bR )原生狀態的脂雙層環境，有助於其在水相之分散能力，並提供其於不同環境調控光迴圈反應之能力。為研究細菌視紫質於不同尺寸環化脂質奈米碟之光迴圈動力學，吾人將去原生脂質之單體細菌視紫質埋入由不同長度環化膜支架蛋白( circularized membrane scaffold protein, cirMSP )所建構的環化脂質奈米碟，並利用尺寸排除層析法驗證其尺寸之差異。此外吾人亦製備原生三聚體細菌視紫質(亦稱紫膜)及界面活性劑微胞( micelle )環境的單體細菌視紫質，以探討化學環境對細菌視紫質光迴圈動力學的影響。而動力學實驗結果顯示奈米碟有效地模擬了細菌視紫質之原生環境，有助於細菌視紫質保有了與紫膜相似的光迴圈活性，如M及O態的瞬態佈居分布。此外，質子化席夫鹼與Asp-85之靜電交互作用為影響M態生成之其中一項因素，而由穩態吸收光譜及動力學實驗結果可知，質子化席夫鹼與Asp-85之靜電交互作用與細菌視紫質之寡聚狀態有關，故三聚體與單體具有不同之M態生成動力學。另一方面，脂質特性及脂質奈米碟尺寸皆有可能造成光迴圈動力學的差異，依照吾人目前的實驗結果未能區別兩種因素的貢獻，故無法單獨指出脂質奈米碟尺寸對光迴圈動力學的影響。然而，本論文仍提供重要的實驗數據以說明單體和三聚體細菌視紫質的光迴圈活性之差異。

Lipid nanodiscs provide bacteriorhodopsin (bR) excellent aqueous dispersibility and native-mimic lipid bilayer environment and are capable of altering bR’s photocycle reaction in various lipid environments. To study the lipid nanodiscs size effect on the photocycle kinetics, the delipidated monomeric bR is embedded into circularized lipid nanodics constructed by different lengths of covalently circularized membrane scaffold proteins (cirMSP). To compare the photocycle kinetics in different chemical environments, the purple membrane (PM) and the monomeric bR reconstituted in different detergent micelles are also prepared. The kinetics experimental results, in terms of transient population of M and O intermediates, reveal that circularized lipid nanodiscs effectively mimic the native environments of PM, assisting bR in preserving the original photocycle activity. On the basis of the steady-state absorption specta and M-rise temporal profile, I proposed that the interaction between protonated Schiff base and Asp-85 is associated with the aggregation state of bR in terms of trimeric and monomeric conditions. However, the properties of lipid and the size of lipid nanodiscs are not distinguishable from each other to contribute the difference in kinetics. Although, I am not able to exclusively conduct the lipid nanodiscs size effect on the photocycle kinetics, this study still provided important results that the photocycle kinetics strongly depends on trimeric and monomeric status of the bR.

第一章 緒論........................................................................................................ 1
1.1 前言........................................................................................................ 1
1.2 文獻回顧................................................................................................ 1
1.2.1 細菌視紫質之發現 ............................................................................... 1
1.2.2 光迴圈及質子幫浦 ............................................................................... 1
1.2.3 細菌視紫質與紫膜的結構 ................................................................... 2
1.2.4 細菌視紫質之單體化 ........................................................................... 2
1.3 研究動機與目的 ................................................................................... 3
1.4 參考文獻................................................................................................ 5
第二章 細菌視紫質與脂質奈米碟之特性 ....................................................... 8
2.1 細菌視紫質相關性質 ........................................................................... 8
2.1.1 細菌視紫質結構及紫膜的組成 ........................................................... 8
2.1.2 靜態紫外/可見光吸收光譜特性 .......................................................... 8
2.1.3 圓二色可見光吸收光譜特性 ............................................................... 9
2.2 細菌視紫質之光迴圈與瞬態吸收光譜性質 ..................................... 10
2.3 質子幫浦與周圍化學環境之關係 ..................................................... 11
2.4 奈米碟之特性與發展 ......................................................................... 12
2.5 參考文獻.............................................................................................. 20
第三章 儀器原理、實驗系統架設與樣品製備 ............................................. 24
3.1 儀器原理.............................................................................................. 24
3.1.1 穩態紫外/可見光吸收光譜法 ............................................................ 24
v
3.1.2 圓二色光譜法 ..................................................................................... 26
3.1.3 快速蛋白質液相層析儀 ..................................................................... 27
3.1.4 可見光瞬態吸收光譜法 ..................................................................... 28
3.2 實驗系統.............................................................................................. 28
3.2.1 穩態紫外/可見光吸收光譜儀 ............................................................ 28
3.2.2 單波長可見光瞬態吸收光譜法 ......................................................... 29
3.2.3 實驗儀器之參數設定 ......................................................................... 30
3.2.3.1 穩態紫外 /可見光吸收譜儀 可見光吸收譜儀 ................................ .............. 30
3.2.3.2 圓二色可見光吸收譜儀 圓二色可見光吸收譜儀 ................................ ................... 30
3.2.3.3 單波長可見光瞬態吸收譜法 單波長可見光瞬態吸收譜法 ................................ ........... 30
3.3 樣品製備.............................................................................................. 31
3.3.1 嗜鹽古菌Halobacteria salinarum的培養 ........................................ 31
3.3.2 紫膜之製備 ......................................................................................... 31
3.3.3 分散於Triton X-100微胞中之單體細菌視紫質之製備 ................. 32
3.3.4 分散於CHAPSO微胞中之去原生脂質單體細菌視紫質之製備 ... 33
3.3.5 環化脂質奈米碟環境之去原生脂質單體細菌視紫質之製備 ......... 33
3.3.6 實驗之樣品種類與溶液環境 ............................................................. 35
3.4 參考文獻.............................................................................................. 55
第四章 實驗結果與討論 ................................................................................. 56
4.1 穩態紫外/可見光吸收光譜 ................................................................ 56
4.2 圓二色光譜 ......................................................................................... 56
4.3 陰離子交換層析圖譜 ......................................................................... 57
vi
4.4 尺寸排除層析圖譜 ............................................................................. 57
4.5 雷射重覆頻率測試 ............................................................................. 58
4.6 動力學實驗 ......................................................................................... 58
4.7 參考文獻.............................................................................................. 69
第五章 結論...................................................................................................... 71

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