李以仁Lee, I-Ren林辰蔓Lin, Chen-Man2023-12-089999-12-312023-12-082022https://etds.lib.ntnu.edu.tw/thesis/detail/3c11b0dbda2476f6c44bc736192d866c/http://rportal.lib.ntnu.edu.tw/handle/20.500.12235/120940鈉離子依賴性膽酸轉運蛋白 (Apical sodium-dependent bile acid transporter; ASBT) 利用鈉離子濃度梯度來驅動運輸膽酸 (bile acid)，在腸肝循環中扮演著回收膽酸的重要角色。當前開發藥物以抑制 ASBT 功能使其減少膽酸的回收，進而降低膽固醇水平被視為具有治療高膽固醇血症(hypercholesterolemia)的潛力。目前對於 ASBT 的了解僅有三種已知的靜態晶體結構，分別為一個與兩個鈉離子及受質結合的向內開口型 (inward-facing)，兩個沒有任何鈉離子及受質結合的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 的構型轉變。我們在 1:20000 蛋白脂質體的實驗中觀察到在三種條件(鉀離子；鈉離子；鈉離子與牛黃膽酸)下 EFRET 值的分布變化與交替通透機制 (alternating access mechanism) 的推測相近。透過分析不同條件下的跳動種類可以發現，鉀離子以頻繁跳動為主要的行為模式，可進一步證明 ASBT 會在兩種構型之間做轉換，但可能存在著一道屏障；而在鈉離子與同時含有牛黃膽酸的情況下則是以傾向於向內開口為主要的行為模式，此現象可以證明鈉離子與牛黃膽酸主導著構型轉變關鍵，這些研究結果提供了 ASBT 更詳細的運輸機制。Sodium-dependent bile acid transporter (ASBT), which utilizes concentration gradient of sodium cation to transport bile acid, plays an important role in the recovery of bile acid in the enterohepatic circulation. It was proposed that the recycling of bile acids, gets retarded when the function of ASBT was inhibited; thereby reducing cholesterol levels. Thus, ASBT is regarded as a target for the development of drugs for the treatment of hypercholesterolemia. Currently, only three known crystal structures of ASBT were identified. Inward-facing structure binding with two sodium cations and substrate bound was found in ASBT homolog from Neisseria meningitides (ASBTNM), and Apo structure with inward- and outward-facing were found in ASBT homolog from Yersinia frederiksenii (ASBTYF). However, these static structures are not sufficient to provide a complete transport mechanism. We utilized single-molecule fluorescence resonance energy transfer (smFRET) microscopy to study the conformational transition of ASBT in micelles and proteoliposomes. In the experiment of 1:20000 proteoliposomes, we observed that the distribution of FRET under the three conditions is similar to the speculation of the alternating access mechanism, which provides a more detailed transport mechanism for ASBT.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Apical sodium-dependent bile acid transporter (ASBT)enterohepatic circulationhypercholesterolemiasingle-molecule fluorescence resonance energy transfer (smFRET)etd