| Summary: | 碩士 === 國立臺灣大學 === 化學工程學研究所 === 102 === 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.
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