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研究生:黃博裕
研究生(外文):Po-Yu Huang
論文名稱:以核磁共振光譜儀探測人類γD水晶體蛋白之結構穩定性
論文名稱(外文):Probing the Structural Stability of Human γD-Crystallinby NMR Spectroscopy
指導教授:王勝仕
指導教授(外文):Sheng-Shih Wang
口試委員:吳宛儒林達顯王孟菊
口試委員(外文):Wan-Ru WuTa-Hsien LinMeng-Jiy Wang
口試日期:2014-07-18
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:化學工程學研究所
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2014
畢業學年度:102
語文別:中文
論文頁數:121
中文關鍵詞:白內障水晶體蛋白人類γD水晶體蛋白化學變性核磁共振光譜
外文關鍵詞:cataractcrystallinhuman γD-crystallinequilibrium chemical unfoldingnuclear magnetic resonance spectroscopy
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白內障(cataract),為全世界人類主要的致盲疾病,尤其好發於老年人。白內障的成因為水晶體中的水晶體蛋白(crystallin)產生聚集,並形成不溶性沉澱物所致。此沉澱物不僅影響水晶體的透明度,並且也改變了水晶體的光折射率,最後導致視力損傷,以至於致盲。目前並無有效治療白內障的藥物,主要醫療途徑仍不免手術植入人工水晶體,但也只有在白內障造成的視力損傷嚴重影響到日常生活時,才會進行手術。
人類γD水晶體蛋白(human γD-crystallin, hgdc),含有174個殘基和兩個結構域(各自由兩個希臘鑰模體組成),兩結構域間由特定殘基側鏈的疏水作用力來穩定,此特定殘基在N端和C端結構域分別為Met44、Phe57、Ile82和Val132、Leu145、Val170。人類γD水晶體蛋白存在於人類眼睛水晶體中作為結構蛋白,對於維持水晶體的透明性扮演著重要的角色。當人類γD水晶體蛋白結構發生改變時,則可能會產生蛋白質聚集現象,並造成視力損失,即是白內障。
本研究中,我們自大腸桿菌BL21(DE3)表現人類γD水晶體蛋白,並經由純化獲得純人類γD水晶體蛋白溶液。接著以1 M胍鹽酸變性劑操作並搭配核磁共振光譜(nuclear magnetic resonance spectroscopy)技術,試圖了解在原態與1M胍鹽酸變性環境下,人類γD水晶體蛋白的殘基、二級結構以及三級結構的差異性。
經由化學位移偏移量、化學位移索引法(chemical shift index)、J偶合預測氫鍵分布、弛緩、三級結構建模等分析,得到結論如下。1 M胍鹽酸變性劑無法使人類γD水晶體蛋白的二級、三級結構展開,僅有影響局部殘基。而在殘基區域D39 ~ C42以及R59 ~ Y63處,受到1 M胍鹽酸變性劑影響相對顯著,此區域乃人類γD水晶體蛋白兩結構域參與界面作用力的區域之一。藉由人類γD水晶體蛋白3D結構的建立與分析,推測在N端和C端結構域之間的界面區域,具有一前一後往復之運動模式。本研究之成果對於人類γD水晶體蛋白的結構不穩處與白內障發生機制間的關聯性,將有更進一步的認識。


Cataract, prevalent among elders, is the main human disease causing blindness in the world. It is believed that the aggregation of crystallin in the human lens is the cause of cataract. The resulting precipitation not only affects the transparency, but also changes the refractive index of human lens, and in the end impairment of vision comes about.Nowadays no effective medicine can cure cataract, and the main operation is the replacement of the catarctous lens with artificial intraocular lens (IOLs), which is patient-unfriendly.
Human γD-crystallin (abbreviated as hgdc in the following text), a 20.6 kDa, 174-residues protein with two domains having two Greek-key motifs respectively, acts as structural protein in human lens and plays an important role in maintaining lens’ transparency.It was reported that the stability of hgdc lies in the interdomain interface, assisted with the side chain hydrophobic interaction of specific residues, including Met44, Phe57, Ile82 in N-domain and Val132, Leu145, Val170 in C-domain. When the loss of the stability of hgdc takes place, it may cause protein aggregation, vision impairment, and that is cataract.
In this thesis, we express hgdc with E. coli BL21(DE3) and obtain the pure hgdc protein solution after purification. With the denaturation of hgdc by 1 M guanidine hydrochloride and the use of nuclear magnetic resonance spectroscopy (NMR), we try to figure out the difference between the native and 1 M guanidine hydrochloride denatured hgdc, covering from the residues level to second and third structure levels.
With the analyses of chemical shift perturbation, chemical shift index, distribution of hydrogen bonding by J coupling, relaxation, and modeling of third structure, we come to the following conclusion. It is not enough to denature hgdc with 1 M guanidine hydrochloride and the extent of influence only falls in local residues. The region D39 ~ C42 and R59 ~ Y63 is relative significantly affected by 1 M guanidine hydrochloride, which are regions participating in the hydrophobic interaction between two domains of hgdc. The result in the thesis will help understand the relation between the stability of hgdc and the mechanism of forming cataract.


目錄
誌謝 I
摘要 III
Abstract IV
目錄 VI
圖目錄 VIII
表目錄 XIII
第一章 序論 1
第二章 文獻回顧 2
2.1 白內障 2
2.2 水晶體蛋白 5
2.2.1 α水晶體蛋白 5
2.2.2 β/γ水晶體蛋白 6
2.2.3 人類γD水晶體蛋白 12
第三章 材料與方法 22
3.1 儀器 22
3.2 菌株與質體 22
3.3 藥品 23
3.4 溶液配製 24
3.5 實驗步驟 26
3.5.1 人類γD水晶體蛋白表現與純化 26
3.5.2 同位素標記人類γD水晶體蛋白表現與純化 27
3.5.3 核磁共振光譜樣品製備 27
3.5.4 聚丙烯醯胺膠體製備 27
3.5.5 核磁共振光譜實驗 28
第四章 結果與討論 33
4.1 人類γD水晶體蛋白表現與純化最適化測試 33
4.2 人類γD水晶體蛋白變性劑滴定測試 36
4.3 人類γD水晶體蛋白HSQC圖指認 42
4.4 人類γD水晶體蛋白二級結構分析 52
4.5 人類γD水晶體蛋白活動性分析 56
4.6 人類γD水晶體蛋白氫鍵分布分析 59
4.7 人類γD水晶體蛋白弛緩分析 61
4.8 以CS-Rosetta建構人類γD水晶體蛋白之起始三級結構 65
4.9 人類γD水晶體蛋白三級結構微調 80
第五章 結論與建議 90
5.1 結論 90
5.2 建議與未來展望 91
參考文獻 92
附錄 102
附錄A 人類γD水晶體蛋白化學位移指認表(原態環境) 102
附錄B 人類γD水晶體蛋白化學位移指認表(1 M胍鹽酸變性環境) 107
附錄C 人類γD水晶體蛋白RDC表(原態環境,4 %聚丙烯醯胺膠體,鑄膠腔體內徑5.4 mm) 112
附錄D 人類γD水晶體蛋白RDC表(1 M胍鹽酸變性環境,4 %聚丙烯醯胺膠體,鑄膠腔體內徑5.4 mm) 117



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