臺灣博碩士論文加值系統

膜蛋白的拓撲結構提供了多肽鏈如何跨越細胞膜的二維信息，例如跨膜區段的數量和長度以及相對於膜的方向。過去科學家利用電腦預測和許多實驗方法來研究結構未知膜蛋白的拓撲圖，但是這些實驗的方法操作起來耗時耗力，並且難以進行定量分析。在本研究中我們設計了一種新的實驗方法，通過凝膠螢光檢測的定點烷基化Site-directed alkylation monitored by in-gel fluorescence (SDAF)，將膜蛋白的C末端融合EGFP，使融合EGFP的膜蛋白能夠在SDS-PAGE凝膠上產生螢光蛋白條帶。我們使用了巰基高分子聚合物反應試劑mPEG-MAL-5K和暴露在細胞表面的目標蛋白之半胱氨酸共價結合，導致在凝膠上產生5kDa螢光條帶移位。位在細胞質側的半胱氨酸則無法結合，故不會有螢光蛋白條帶的移位。我們使用晶體結構已經確定的膽汁酸轉運蛋白ASBT_NM作為模型，證明SDAF可以產生與晶體結構一致的拓撲圖。此外由於SDAF可以簡單的控制mPEG-MAL-5K與蛋白質的莫耳數比，因此可以用來進行不同位點的半胱氨酸PEGylation程度之定量分析。利用量化分析的結果，我們可以知道不同半胱氨酸的溶劑可及性，並利用SDAF測定每個半胱氨酸被PEGylation程度，進一步用於了解天然細胞膜環境中目標膜蛋白的動態信息，以研究膜蛋白在不同buffer環境下之構型偏好性。

The topology of helix-bundle membrane proteins provides the two-dimensional information of how the polypeptide chain spans the cell membrane: the number and length of transmembrane segments, and the orientation in relative to the membrane. In the past, in silico topology prediction and many experimental methods have been developed to determine the topology of structurally-unknown membrane proteins, but these methods are either time consuming or labor intensive, and difficult to perform quantitative analysis. In this study, we designed a new experimental method, the site-directed alkylation detected by in gel fluorescence (SDAF), using GFP fused to the C-terminus of target membrane proteins to facilitate the imaging of the fluorescent target proteins on SDS-PAGE gels. The thiol-reactive polymer mPEG-MAL-5K can be covalently conjugated to the cysteine residues introduced on the extracellular loops of the target protein by site-directed mutagenesis, resulting in a 5 kDa band shift on the gel. On the contrary, the cysteines introduced on the intracellular loops cannot be PEGylated, and thus resulted in no band shift. We used the structurally determined bile acid transporter ASBTNM as a model, and demonstrated that SDAF can produce a topology map consistent with the crystal structure. Moreover, as the mPEG-MAL-5K to protein molar ratio can be constantly maintained, one can perform a quantitative analysis of the PEGylation level, which reflects the solvent accessibility of a given cysteine residue. A systematic SDAF assay of cysteine replacements generates a PEGylation profile which can be further employed to provide dynamic information of membrane proteins in native cell membrane environments, by which the conformation preference of the membrane proteins in different buffer conditions can be probed.

目錄
中文摘要 i
Abstract ii
目錄 iii
圖表目錄 vi
1. 前言 1
1.1. 二級主動轉運蛋白 1
1.1.1. 細胞膜上的蛋白 1
1.1.2. 二級主動轉運蛋白的機制 1
1.2. 交替通透機制(Alternating Access Mechanism) 2
1.2.1. 跨膜蛋白轉運受質時的機制 2
1.2.2. 生化實驗與蛋白質晶體學發現不同交替通透機制模型 2
1.3. 鈉離子依賴性膽酸轉運蛋白ASBT 3
1.3.1. Solute carrier superfamily (SLC family) 3
1.3.2. 膽酸循環與ASBT的重要性 3
1.4. ASBT的蛋白晶體構型 4
1.4.1 ASBT_NM 晶體結構 4
1.4.2. ASBT_YF 晶體結構 4
1.4.3. ASBT轉運受質時產生的elevator-like movement 5
1.5. 研究拓撲來得到膜蛋白的資訊 5
1.5.1. 膜蛋白結晶的困難 5
1.5.2. 電腦拓撲預測 5
1.5.3. 實驗方法 6
1.6. 本論文研究目的 6
2. 實驗與方法 7
2.1. ASBT_NM突變株 7
2.1.1. 決定拓撲的ASBT_NM單點突變 7
2.1.2. 位點導向基因突變(Site-Directed Mutagenesis) 8
2.1.3. DNA膠體電泳 9
2.1.4. Competent cell製備 9
2.1.5. 轉型作用(transformation) 10
2.1.6. 抽質體DNA 10
2.2. Site-Directed Alkylation Detected by In-Gel Fluorescence (SDAF) 11
2.2.1. cfASBT_NM-EGFP 和突變株的表達 11
2.2.2. 使用mPEG-MAL-5K標記E. coli whole cells 11
2.2.3. 使用mPEG-MAL-5K標記E. coli之破菌後細胞膜 12
2.2.4. N-Ethylmaleimide (NEM) blocking 13
2.2.5. 使用凝膠內螢光成像(Imaging using in-gel fluorescence) 13
2.2.6. 量化PEGylation的程度 14
2.3. WT及突變株ASBT_NM蛋白的膽酸運輸功能性測試 14
2.3.1. WT及突變株ASBT_NM-EGFP 的蛋白表達 14
2.3.2. 標準化細胞量 14
2.3.3. 運輸功能性測試 15
3. 結果 17
3.1. SDAF應用於膜蛋白拓撲學 17
3.1.1. mPEG-MAL-5K可標記ASBT_NM在E. coli內膜外側loop上之半胱氨酸 17
3.1.2. E. coli破菌後細胞膜可利用mPEG-MAL-5K標記細胞內側的半胱氨酸 17
3.1.3. 檢查EGFP中兩個內源性半胱氨酸的mPEG-MAL-5K溶劑可及性 17
3.1.4. 用已知結構ASBT_NM驗證SDAF決定膜蛋白拓撲的可行性 18
3.1.5. ASBT_NM 半胱氨酸被PEGylation的程度反映該位點的溶劑可及性 19
3.2. WT及突變株ASBT_NM蛋白的膽酸運輸功能性測試(uptake assay) 19
3.2.1. 標準化[³H]-taurocholate運輸功能 19
3.2.2. 決定topology的ASBT_NM單點突變株功能性測試 20
3.2.3. 受質運輸途徑的ASBT_NM單點突變株功能性測試 20
4. 討論 21
4.1. 膜蛋白拓撲學結構的測定技術 21
4.1.1. 利用C端酵素融合決定膜蛋白拓撲結構 21
4.1.2. Substituted cysteine accessibility method (SCAM™) 21
4.1.3. SDAF應用於決定膜蛋白拓撲結構 22
4.2. SDAF應用於膜蛋白拓撲學的研究之考量與限制 22
4.2.1. EGFP的突變株並不會影響蛋白折疊 22
4.2.2. 細胞內PEGylation程度較低 23
4.2.3. 在破菌條件中沒有free GFP的訊號 23
4.2.4. EGFP中兩個內源性半胱氨酸對SDAF的影響 24
4.3. 用SDAF定量分析的優勢 24
4.3.1. SDAF可以進行定量分析 24
4.3.2. 使用Western blot進行定量分析之難處 24
4.3.3. SDAF可以看膜蛋白動力學變化 25
4.3.4. 目前已知研究膜蛋白動力學變化之方法 25
4.4. 結論 25
5. 圖表 26
6. 參考文獻 54

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