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研究生: 古芝綺
Ku, Chih-Chi
論文名稱: 利用單分子螢光共振能量轉移光譜觀察鈉離子依賴性膽酸轉運蛋白(ASBT)的動態變化
Conformational Dynamics of the Apical Sodium-dependent Bile acid Transporter (ASBT) Studied by Single-Molecule Fluorescence Resonance Energy Transfer Spectroscopy
指導教授: 李以仁
Lee, I-Ren
口試委員: 胡念仁
Hu, Nien-Jen
孫英傑
Sun, Ying-Chieh
口試日期: 2021/08/17
學位類別: 碩士
Master
系所名稱: 化學系
Department of Chemistry
論文出版年: 2021
畢業學年度: 109
語文別: 中文
論文頁數: 48
中文關鍵詞: 鈉離子依賴性膽酸轉運蛋白(ASBT)腸肝循環高膽固醇血症單分子螢光共振能量轉移
英文關鍵詞: Apical sodium-dependent bile acid transporter(ASBT), enterohepatic circulation, hypercholesterolemia, single-molecule fluorescence resonance energy transfer(smFRET)
DOI URL: http://doi.org/10.6345/NTNU202101169
論文種類: 學術論文
相關次數: 點閱:106下載:1
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  • 鈉離子依賴性膽酸轉運蛋白(Apical sodium-dependent bile acid transporter; ASBT)利用膜內外的鈉離子濃度梯度驅動運輸膽酸(bile acid),被認為是腸肝循環(enterohepatic circulation)中膽酸回收的第一步。抑制ASBT功能會減少膽酸的回收,導致膽酸的合成增加和降低膽固醇,可用於治療高膽固醇血症(hypercholesterolemia)。目前已有三種ASBT晶體結構被解出,分別為含有兩個鈉離子與一個受質結合的向內開口型(inward-facing),與沒有任何離子與受質的結合時,向內開口型及向外開口型(outward-facing)。然而,這些靜態結構不足以提供一個完整的運輸機制,我們利用單分子螢光共振能量轉移光譜觀察蛋白脂質體與微胞中ASBT的構型動力學。我們在實驗中觀察到可互相轉換的三種構型,其狀態轉變之動力學受溶液中的離子與膽酸濃度影響,可以提供膜蛋白更詳細的運輸機制。

    Apical sodium-dependent bile acid transporter (ASBT) utilizes sodium gradient to transport bile acid and is considered the first step for bile acid reabsorption in the enterohepatic circulation. Inhibiting the ASBT function reduces the reabsorption of bile acid synthesized from cholesterol. Inhibition of ASBT promotes the synthesis of bile acid and reduction of cholesterol for the treatment of hypercholesterolemia. Three crystal structures of ASBT have been reported: Inward-open structure binding with bile acid taurocholate and two sodium cations was found in ASBT homolog from Neisseria meningitides (ASBTNM). Apo structures with inward- and outward-open structures were resolved in ASBT homolog from Yersinia frederiksenii (ASBTYF). However, these static structures are insufficient to provide a comprehensive transportation mechanism, which is expected to be highly dynamic. We utilize single-molecule fluorescence resonance energy transfer (smFRET) microscopy to observe the direct conformation dynamics in the micelle and proteoliposome. Our result revealed that there are transitions between three major conformations. Cation- (K+ and Na+), as well as the cargo- (taurocholate in Na+), dependent transition kinetics was observed. Thus, the direct observation of conformational change can provide more detailed information on the transport mechanism.

    第一章、緒論 1 1-1 前言 1 1-2 膽酸的腸肝循環(enterohepatic circulation) 2 1-3 ASBT的相關疾病與治療 3 1-4 交替通透機制(alternating access mechanism) 4 1-5 ASBT的結構 6 1-6 研究動機 9 第二章、實驗儀器與方法 10 2-1 實驗原理與儀器 10 2-1-1 單分子螢光共振能量轉移 10 2-1-2 全內反射螢光顯微鏡 14 2-2 實驗樣品的製備 16 2-2-1 實驗用玻片表面修飾 16 2-2-2 實驗用玻片組裝 19 2-2-3 實驗樣品的設計 20 2-2-4 實驗樣品的固定 22 2-2-5 顯影緩衝溶液(image buffer) 23 2-3 數據處理與分析 26 第三章、實驗結果與討論 30 3-1實驗設計 30 3-2 ASBT在蛋白脂質體的分析 31 3-2-1 EFRET分布直方圖 31 3-2-2 長時間軌跡圖 33 3-2-3 蛋白脂質體固定問題 37 3-3 ASBT在微胞的分析 38 3-3-1 EFRET分布直方圖 38 3-3-2 長時間軌跡圖 39 3-3-3 ASBT在微胞中的構型轉換 41 第四章、結論與未來展望 43 參考文獻 44

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