臺灣博碩士論文加值系統

δ-晶體蛋白為鳥類和爬蟲類的水晶體特有之結構蛋白，與精胺醯丁二酸裂解酶(argininosuccinate lyase, ASL)有高度序列相似性，具演化關係。先前研究將δ-晶體蛋白位於單元介面間上的K315突變成Ala時，在尿素影響下，以色胺酸螢光追蹤蛋白質結構變化，發現存在穩定單體的中間狀態，而野生型則無此現象。本研究進一步想了解此中間狀態結構，可逆性變性及單元組合過程。野生型與突變型為3階段變性過程，所需消耗能量分別為9.9 ± 0.4 (kcal/mol)及8.6 ± 0.3 (kcal/mol)，推論K315在維持單元介面上之安定能量約為1.3 (kcal/mol)。K315A在尿素環境下形成之中間狀態其二級結構與原來結構沒有顯著差異，而此狀態較原來結構有較高之拒水性區暴露但不形成聚合物結構，可能為單元解離後形成穩定單元體。當稀釋尿素之濃度後，K315A可由此中間狀態結構再折疊成四聚體。但尿素變性後之δ-晶體蛋白結構卻無法可逆性恢復。K315A穩定中間狀態，在胍胺酸作用，可以可逆性以3 – state方式變性及復性，其能量為3.1(kcal/mol)。此結果，符合先前推測δ-晶體蛋白可逆性變性過程T<-->D<-->M<-->I1<-->U。而K315在單元介面之作用力推測為造成蛋白質單元解離過程之能量障礙，因此野生型δ-晶體蛋白之單元解離與部分結構瓦解同時進行，而形成部分變性之單元體結構。

δ-crystallin is a taxon-specific crystallin that expressed in the eye lenses of birds and reptiles .In evolution, birds, δ-crystallin was recruited from the housekeeping enzyme argininosuccinate lyase (ASL) and serve as a structure protein. Conformation change of δ-crystallin was studied previously by using tryptophan fluorescence as probe. A stable intermediate was found during unfolding of K315A mutant but not for wild-type δ-crystallin. K315 forms impatail H-bonding between subunit interfaces. To elucidate the structure role of k315, the reversible unfolding and association of the stable intermediate was studied. Unfolding of wild-type and mutant δ-crystallin by urea follows 3 – state process. The total energy for unfolding were 9.9 ± 0.4 and 8.6 ± 0.3 (kcal/mol), respectively. Therefore, the energy for K315 to maintain the stability of interface was about 1.3 (kcal/mol). No apparent secondary structure changes was observed between the stable intermediate and native form. However, there was a higher exposure of the hydrophobic area. But no aggregation was found. Thus the stable intermediate suggested to be a stable monomer. When the urea was removed, monomer K315A was able to reassociated into a tetramer. However, the association of partial unfolded wild-type intermediate form was irreversible unfolding monomeric under GdnHCl was reversible following a 3 - state unfolding models with the unfolding energy about 3.1 kcal/mol. The result was consistent with our previous assumption that the reversible unfolding of δ-crystallin follows the process of T<-->D<-->M<-->I<-->U. The interaction provided by K315 in subunit interface is the energy barrier for subunit dissociated. Consequently, the simultaneously occurrence of dissociate and unfolding for wild-type δ-crystallin in urea led to the formation of partial unfolded intermediate.

總目錄
目錄-----------------------------------------------II
表目錄----------------------------------------------IV
圖目錄----------------------------------------------V
附錄目錄--------------------------------------------VII
名詞縮寫對照表---------------------------------------VIII
摘要-----------------------------------------------IX
Abstract------------------------------------------X

目錄
緒論-------------------------------------------------01
實驗材料----------------------------------------------05
壹、 藥品試劑--------------------------------------05
貳、 儀器及器材------------------------------------06
參、 試劑配製--------------------------------------07
實驗方法----------------------------------------------11
壹、 勝任細胞 (DH5α、BL21)製作----------------------11
貳、 轉殖作用 (Transformation)---------------------11
參、 野生型與突變型蛋白質之菌體培養--------------------12
肆、 野生型與突變型之蛋白質純化-----------------------13
伍、 測定蛋白質濃度---------------------------------16
陸、 圓偏振二色旋光光譜儀之蛋白質二級結構分析------------17
柒、 尿素或GdnHCl效應下之變性及復性實驗----------------17
捌、 蛋白質色胺酸螢光之實驗---------------------------18
玖、 ANS螢光實驗分析--------------------------------18
壹拾、 分析級超高速離心實驗-----------------------------19
壹拾壹、 分子過篩層析實驗--------------------------------20

實驗結果----------------------------------------------22
壹、 野生型δ-和突變型δ-晶體蛋白之純化------------------22
貳、 尿素效應下的變性實驗----------------------------23
參、 尿素效應下蛋白質單元解離之熱力學常數分析------------23
肆、 ANS結合分析-----------------------------------24
伍、 圓偏振二色旋光光譜分析---------------------------24
陸、 分析型超高速離心分析-----------------------------25
柒、 尿素變性後之復性--------------------------------26
捌、 GdnHCl效應下之變性實驗--------------------------26
討 論----------------------------------------------28
參考文獻-----------------------------------------------53

表 目 錄
表一、野生型及K315A突變型δ-晶體蛋白在尿素變性實驗之熱力學
參數分析-----------------------------------------45
表二、不同尿素濃度下野生型及突變型δ-晶體蛋白二級結構分析-
-----------------------------------------------46
表三、將不同尿素之濃度稀釋到0.15M野生型及突變型δ-晶體蛋白
二級結構分析-------------------------------------46
表四、K315A突變型δ-晶體蛋白單元體在GdnHCl變性實驗之熱力學
參數分析-----------------------------------------47

圖 目 錄
圖一、野生型鵝眼δ-晶體蛋白立體結構-------------------------32
圖二、野生型鵝眼δ-晶體蛋白純化圖---------------------------33
圖三、K315A突變型鵝眼δ-晶體蛋白純化圖----------------------34
圖四、野生型及突變型δ-晶體蛋白尿素變性實驗-------------------35
圖五、野生型及突變型鵝眼δ-晶體蛋白ANS結合分析----------------36
圖六、野生型δ-晶體蛋白在不同尿素濃度下蛋白質圓偏振二色旋光
譜----------------------------------------------37
圖七、K315A突變型δ-晶體蛋白在不同尿素濃度下蛋白質圓偏振二
色旋光譜-----------------------------------------38
圖八、野生型及突變型δ-晶體蛋白在不同尿素濃度下二級結構之變
化---------------------------------------------39
圖九、超高速離心分析野生型及K315A突變型δ-晶體蛋白在不同尿
素濃度下蛋白分子分布情形---------------------------40
圖十、野生型及K315A突變型δ-晶體蛋白在不同尿素變性後之復性
實驗分析----------------------------------------41
圖十一、野生型與K315A突變型δ-晶體蛋白變性後復性實驗分析-
------------------------------------------------------42
圖十二、K315A突變型δ-晶體蛋白單元變性分析-------------------43
圖十三、K315A突變型δ-晶體蛋白單元變性後復性分析--------------44

附錄目錄
附錄一、模擬鵝眼δ-晶體蛋白的活性中心------------------------48
附錄二、模擬鵝眼δ-晶體蛋白胺基酸K315在兩雙聚體介面間氫鍵形
成-------------------------------------------49
附錄三、突變型鴨眼δ-晶體蛋白S283A與野生型鴨眼δ-晶體蛋白活
性中心內參與受質催化的胺基酸經結構重疊之結果-
----------------------------------------------50
附錄四、野生型鵝眼δ-晶體蛋白單元與單元間作用力----------------51
附錄五、突變型鵝眼δ-晶體蛋白K315A 單元與單元間作用力
---------------------------------------------52

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