臺灣博碩士論文加值系統

小腸頂區鈉離子依賴性膽酸轉運蛋白(Apical Sodium-dependent Bile acid Transporter, ASBT)，是一個位在小腸頂區絨毛上皮細胞上，藉由鈉離子濃度梯度，來運送受質膽酸的二級主動運輸蛋白。ASBT主要功能是回收腸道中的膽酸，透過腸肝循環後運回到肝細胞當中。過去關於蛋白結構的研究利用細菌同源蛋白 ASBTNm得到受質結合口袋向內之構型之晶體結構，與另一個細菌同源蛋白ASBTYf受質結合口袋向外與受質結合口袋向內的兩個構型之結構。經由這三個晶體結構在構型上的快照圖，可以建立出一套Elevator-like的受質運輸機制模型。但由於缺乏ASBT構型在不同環境下改變方式與動態上的資訊，運輸機制仍無法完全確立。在本實驗室先前的研究中，我們開發了SDAF(Site-directed Alkylation monitored by in gel Fluorescence)這項技術，以in situ的方式觀察 ASBTNm 在不同環境下受質結合口袋向外界溶劑通透性的變化。本篇我們也嘗試將SDAF這項技術應用於測定未知結構膜蛋白的拓樸學結構，我們以已知結構的膜蛋白ASBTNm進行測定的結果完全符合我們的預期，證明這項應用是可行的。並且為了測定先前SDAF研究中，一系列受質通道上胺基酸點突變為cysteine的突變株其功能是否受到影響，我們將這些突變株分別在E. coli C43細胞中進行過表現，並以輻射標定的受質[3H]-taurocholate來測量這些突變株在細胞上的功能性。雖然大部分突變株功能性都較WT的ASBTNm還低，但都還是保有運輸受質的功能性。並且為了證實先前SDAF研究中ASBTNm 在不同環境下構型改變動態上的資訊，我們自旋標定(spin-label)了ASBTNm 上的Y39 和 T303並進行EPR (Electroparamagnetic Resonance)光譜學的方法與DEER (Double Electron-Electron Resonance)量測這兩個氨基酸的距離分佈。結果發現在鉀離子環境下ASBTNm蛋白分子主要處於兩種構型分布，分別是inward-facing與outward-facing的構型，而在鈉離子環境下主要為inward-facing的構型，在鈉離子與taurocholate環境下主要為occluded構型。為了取得ASBTNm在雙層磷脂質上的蛋白樣品以供DEER實驗偵測，我們也嘗試以SMALP(styrene maleic acid lipid particles)純化ASBTNm-GFP的融合蛋白，但結果純度不佳，表示這個純化方式還需要進一步的優化。此外我們也嘗試以APBS表面帶電性分析與SwissDock受質結合位預測ASBTNm可能的受質結合位，預測的結果與我們預期相符合。此研究對於未來得到與膽酸結合的outward-facing構型結構提供可能的方法。

Apical Sodium-dependent Bile acid Transporter (ASBT), a Na+-dependent secondary active transporter, is localized at the apical membrane of enterocytes and mediates the reuptake of bile acid from ileum back to liver. The structural information of ASBT is attributed to the crystal structures of bacterial homologs ASBTNm and ASBTYf, revealing the snapshots at inward- and outward-facing states, and the putative mechanism of substrate translocation via an “elevator-like” movement. Nevertheless, the triggers of conformational changes and the dynamics at different substrate conditions remain undetermined. In this project we employed Site-directed Alkylation monitored by in gel Fluorescence (SDAF) to probe the in situ alteration of substrate binding pocket accessibility. We tried to apply SDAF assay in topology determination of membrane proteins, and the results demonstrated it is valid and much more efficient than previous methods. In our earlier SDAF studies, a series of residues located at the substrate pathway in ASBTNm were substituted with cysteine, but the functional effects of these point mutations are still unknown. We performed whole cell [3H]-taurocholate uptake assay using ASBTNm-overexpressed E. coli C43 to monitor the transportation activities of these mutants. Although most ASBTNm mutants have lower activities than WT ASBTNm, they still reserve transport activity at certain level. In order to validate our dynamics data obtained by SDAF, we spin-labelled Y39 and T303 for Double Electron-Electron Resonance (DEER) using Electroparamagnetic Resonance (EPR) spectroscopy. The DEER results indicated that DDM-solubilized ASBTNm is populated as two discrete conformational species in K+ buffer, representing outward- and inward-facing states, and one major conformational species in Na+ buffer, and in taurocholate added Na+ buffer, representing inward-facing and occluded states. In order to obtain soluble ASBTNm in lipid bilayer for DEER spectroscopy, we tried to purify ASBTNm in styrene maleic acid lipid particles (SMALP). The C-terminal GFP fusion facilitates tracking of ASBTNm-SMALP, although the purity requires further optimization. The soluble membrane protein particles in SMALP will enable us to preserve the native behavior of ASBTNm for structural and dynamical studies. We also analized surface charge of ASBTNm using APBS, and performed SwissDock for prediction of possible substrate binding sites. The predicted results are of our expectation. The studies will provide practical methods in obtaining crystal structure of ASBTNm bound with taurocholate at outward-facing state.

中文摘要 I
英文摘要 II
目錄 III
圖表目錄 V
1. 研究背景介紹 1
1.1. 前言 1
1.2. 二級主動轉運蛋白 2
1.3. 交替通透機制(Alternating Access Mechanism) 2
1.4. 三種不同的交替通透機制模型 3
1.5. 鈉離子依賴性膽酸轉運蛋白ASBT (Apical Sodium-dependent Bile acid Transporter) 5
1.6. 研究主軸與動機 7
2. 實驗與方法 9
2.1. 點突變 9
2.2. Site-Directed Alkylation monitored by in gel Fluorescence (SDAF) 10
2.3. 脈衝雙電子偶極耦合DEER (Double Electron-Electron Resonance) 13
2.3.1. ASBTNm Membrane製備 14
2.3.2. ASBTNm之純化 15
2.3.3. Spin label 16
2.4. ASBTNm蛋白與突變蛋白的膽酸運輸功能性測試: 17
2.5. SMA (styrene maleic acid ) 18
3. 結果 20
3.1. SDAF應用於膜蛋白拓樸學的測試 20
3.2. ASBTNm突變株的功能性測試(uptake assay) 21
3.3. 藉由DEER的技術觀察ASBTNm-CS-Y39C-T303C在不同離子條件下的構型動態 22
3.4. 以SMA進行純化ASBTNm-WT-GFP 23
3.5. ASBTNm受質結合口袋位置預測與序列比對 24
3.5.1. ASBTNm晶體結構表面帶電荷分析 25
3.5.2. 利用SwissDock網站計算自由能推測ASBTNm可能的受質結合位置 26
3.5.3. ASBT序列比對 27
4. 討論 28
4.1. 膜蛋白拓樸學結構的測定技術 28
4.2. ASBTNm在不同環境下的構型轉變 29
4.3. ASBTNm的受質結合口袋位置 30
4.4. ASBTNm outward open構型晶體結構 31
5. 參考文獻 32

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