以核磁共振光譜儀探測人類γD水晶體蛋白之結構穩定性

Abstract

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.