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研究生： 曾科嘉
Tseng, Ke-Chia
論文名稱： 以溫度躍升法搭配共軛焦螢光擷取系統研究牛血清白蛋白於原生態溫度區間的蛋白質動態過程
Monitoring the Protein Dynamics of Bovine Serum Albumin in the Native Condition with Confocal Fluorescent Temperature Jump
指導教授： 朱立岡
Chu, Li-Kang
口試委員: 洪嘉呈
Horng, Jia-Cherng
李政怡
Lee, Cheng-I
學位類別： 碩士
Master
系所名稱： 理學院 - 化學系
Department of Chemistry
論文出版年： 2019
畢業學年度： 107
語文別： 中文
論文頁數： 96
中文關鍵詞： 圓二色光譜術 、螢光光譜術 、螢光溫度計 、溫度躍升法 、共軛焦螢光擷取系統 、牛血清白蛋白 、色胺酸 、蛋白質動態學 、活化能 、阿瑞尼斯方程式 、升溫速率
外文關鍵詞： Circular dichroism spectroscopy, Fluorescence spectroscopy, Fluorescent thermometer, Temperature-jump method, Confocal fluorescent system, Bovine serum albumin (BSA), Tryptophan, Protein dynamics, Activation energy, Arrhenius equation, Heating rate
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    • 當溫度、壓力、酸鹼值及化學試劑等外在環境改變時，常會誘發蛋白質發生構形改變，而錯誤的構形常會引發疾病的產生，因此蛋白質構形變化的相關研究廣受關注。本篇論文的研究分子，牛血清白蛋白，與人類血清白蛋白同源性高，故常被當作模型蛋白。在過去牛血清白蛋白靜態的熱致變性研究中，皆著重於溫度高於50 °C後的變性結果。然而，於較接近生理溫度範圍的熱致構形改變之動態過程尚未被廣泛探討。因此吾人以一自組裝式空間暨時間解析溫度躍升螢光系統提供100 μm的空間解析度及秒尺度的升溫速率，觀測牛血清白蛋白於不同起始溫度下(25-42 °C)的動態構形改變過程。吾人發現當起始溫度為25 °C時，牛血清白蛋白經5 °C的溫度躍升後其螢光強度變化趨勢與純色胺酸相異，吾人推論係牛血清白蛋白發生構形改變，並以單指數函數擬合後，得上升時間為27±1 s；而當起始溫度高於30 °C時，則無法再觀測到此構形改變的訊號。此外，吾人觀測到以較快的升溫速率進行溫度躍升會影響牛血清白蛋白構形改變途徑的比例，而此觀點解釋了溫度躍升結果與升溫速率較慢之靜態螢光光譜之間的觀測結果之差異。此外，吾人也透過變溫遠紫外光圓二色光譜及變溫靜態螢光光譜觀測牛血清白蛋白的熱致構形改變，二者可分別提供巨觀及局部結構改變的互補資訊。本篇論文證實了以共軛焦螢光擷取系統改良溫度躍升法的可行性，且所用的實驗技術及數據分析方法提供了對於較大蛋白質之動態過程研究上新的觀點。

    Protein conformational change can be initiated by a variety of external conditions, such as changes in temperature, pressure, pH values, and the addition of chemical denaturants. Misfolding or aggregation of proteins may lead to numerous diseases. Hence, unraveling protein dynamics has attracted particular attentions. Bovine serum albumin (BSA) is usually served as the alternative of human serum albumin (HSA) in kinetic and affinity tests with drugs due to its high homology with HSA. A number of studies have concentrated on its thermally-induced protein unfolding or denaturing above 50 °C. However, the dynamic alteration of BSA structure has not been revealed in the physiological condition. In this work, we employed a second-resolved temperature jump apparatus coupled with the confocal fluorescent thermometry of 100 μm spatial resolution to investigate the dynamical processes of BSA at initial temperatures in 25-42 °C. The discrepancies of the evolutions of the tryptophan fluorescence intensity change of BSA and pure tryptophan upon increasing 5 °C from 25 °C referred to an intrinsic protein dynamics which was quantified with an apparent rise time of 27±1 s. Nevertheless, the aforementioned process was not observed at initial temperatures above 30 °C. Besides, a rapid temperature increase may benefit the detouring toward the reaction which possesses a higher apparent activation energy that is not accessible in the steady-state measurements using a slow heating. In addition, the temperature-dependent steady-state circular dichroism and tryptophan fluorescence spectra could reflect the global and localized structural alterations of BSA upon heating, respectively. In this work, we demonstrated the employment of a confocal fluorescent thermometer to precisely monitor the minute detection volume. Moreover, the combination of the experimental approach and concomitant analysis provides a new viewpoint to illustrate the dynamic processes of a big protein upon thermal stimulus.

    第一章 緒論 1 1.1 前言 1 1.2 文獻回顧 1 1.2.1 溫度躍升法 1 1.2.2 牛血清白蛋白 2 1.3 實驗動機與目的 6 1.4 共軛焦顯微成像技術 8 1.5 色胺酸的螢光性質 8 1.5.1 色胺酸的螢光機制 8 1.5.2 色胺酸螢光強度與溫度的相依性 9 1.5.3 蛋白質中色胺酸之螢光 10 第二章 實驗儀器原理與架設 38 2.1 吸收光譜 38 2.2 圓二色光譜 39 2.3 靜態螢光光譜 41 2.4 空間暨時間解析溫度躍升螢光系統 43 第三章 樣品製備與實驗步驟 53 3.1 樣品製備 53 3.2 紫外-可見光吸收光譜偵測 53 3.3 變溫遠紫外光圓二色光譜偵測 54 3.3.1 牛血清白蛋白之變溫遠紫外光圓二色全光譜 54 3.3.2 牛血清白蛋白於222 nm橢圓率之溫度側寫 54 3.4 變溫靜態螢光光譜偵測 54 3.5 空間暨時間解析溫度躍升螢光光譜偵測 54 3.5.1 共軛焦螢光偵測系統收光位置優化 55 3.5.2 以低功率1550 nm連續式雷射提升樣品起始溫度 55 3.5.3 牛血清白蛋白之不同起始溫度之溫度躍升 56 3.5.4 牛血清白蛋白之不同升溫速率之溫度躍升 56 第四章 實驗結果與討論 68 4.1 樣品濃度定量及靜態定溫吸收及螢光光譜分析 68 4.2 空間暨時間解析溫度躍升螢光系統的建置及優化 69 4.2.1 以色胺酸作為螢光溫度計 69 4.2.2 共軛焦螢光偵測系統收光位置優化 70 4.2.3 以低功率1550 nm連續式雷射提升樣品起始溫度 71 4.3 牛血清白蛋白之變溫靜態光譜 71 4.3.1 變溫遠紫外光圓二色光譜 71 4.3.2 變溫靜態螢光光譜 72 4.4 牛血清白蛋白之溫度躍升實驗 72 4.4.1 不同起始溫度之溫度躍升 72 4.4.2 不同升溫速率之溫度躍升 76 第五章 結論 95

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