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研究生：	盧守筠 Lu, Shou-Yun
論文名稱：	利用定點突變研究二價金屬離子在大白鼠αB-水晶體蛋白之鍵結位置 Site-Directed Mutagenesis Studies on the Divalent Metal Ions Binding Site of Rat Lens αB-Crystallin
指導教授：	黃福永 Huang, Fu-Yung
學位類別：	博士 Doctor
系所名稱：	理學院 - 化學系 Department of Chemistry
論文出版年：	2014
畢業學年度：	102
語文別：	英文
論文頁數：	93
中文關鍵詞：	αB-水晶體蛋白、伴護活性、表面疏水性、定點突變、圓二色光譜
外文關鍵詞：	αB-crystalin, Chaperone activity, Hydrophobicity, Site-directed mutagenesis, Circular dichroism.
相關次數：	點閱：67 下載：1
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αB-水晶體蛋白具有分子伴護功能 (chaperone-like function)，具有維持眼球水晶體之透明度，並防止或減輕因外來壓力(foreign stress)及老化所造成的水晶體蛋白質聚集。在非透鏡狀細胞 (nonlenticular cell)，它參與了多種神經性疾病、糖尿病以及癌症。在αB-水晶體蛋白生物學上，一些金屬離子的作用已被報導。在理論計算方面也已指出，人類αB-水晶體蛋白與二價金屬離子的配位環境上其結合的位點有His101、His119 、Lys121、His18以及Glu99。本突變研究顯示，在大白鼠αB-水晶體蛋白中，His18和His119以及H101是銅和鋅離子影響分子伴護活性和結構之關鍵結合位點。在這項研究中，大白鼠αB-水晶體蛋白的突變體H18G、H119G、H101G分別被選殖和表現後，以研究His18和His119以及H101是否為關鍵結合位點。1mM銅和鋅離子明顯增加αB-水晶體蛋白之分子伴護活性，而1mM鋅和銅以及鎂離子卻顯著減少突變型之活性。ANS螢光測量顯示分子伴護活性和表面疏水性之間並非呈現線性關係，這說明表面疏水性不是分子伴護活性之先決條件。遠和近紫外光圓二色光譜顯示，野生型呈現較多的β-折疊結構特徵; 而突變型呈現較多無規則線圈 (random coil) 特性，以及鍵結位置周圍的色氨酸殘基在環境上具有微觀的變化。由分子伴護活性、ANS螢光測量、遠和近紫外光圓二色光譜的研究結果顯示，His18和His119以及His101置換為甘氨酸(Glycine)後會導致構形和環境上微觀之變化，在二價金屬離子存在下則會降低突變型之分子伴護活性，這表示His18和His119以及H101是銅和鋅離子之關鍵結合位點。綜合以上結果指出， His18和His119以及 His101彼此之間的配位環境是銅和鋅離子的結合位位置，並具有提升水晶體/金屬離子複合物的分子伴護活性和穩定性。

The lens αB-crystallin, playing a major role in maintaining the transparency of the eye lens, possesses chaperone-like function to prevent the lens proteins from aggregation due to foreign stress and/or aging. In nonlenticular cells, it is involved in various neurological diseases, diabetes, and cancer. The role of some metal ions in the αB-crystallin biology has been reported. Theoretical calculations have proposed that the coordination site of human αB-crystallin for binding divalent metal ions were His101, His119, Lys121, His18 and Glu99. In this study, H18G, H119G, and H101G mutant types of rat lens αB-crystalin were cloned and expressed to investigate whether His18, His119 and His101 are the coordination binding sites. And the results suggested that His18, His119 and His101 were the crucial binding sites for Cu (II) and Zn (II) in terms of chaperone-like activity and structure. Copper and zinc at 1 mM concentration significantly increase the chaperone-like activity in wild type αB-crystalin, whereas zinc, copper and magnesium at 1 mM reduced the activity of mutant type significantly. ANS fluorescence measurement showed that there was no linear relationship between chaperone-like activity and surface hydrophobicity, indicating that surface hydrophobicity is not prerequisite for exhibiting chaperone-like activity. Both the Far- and Near-UV CD spectra suggested that the wild type reflected more β-sheet structural characteristics; whereas the mutant type reflected more random coil characteristics and more micro-environmental changes around the tryptophan residues. The results from chaperone-like activity, ANS fluorescence measurement and Far- and Near-UV CD studies indicate that the replacement of His18, His119 and His101 with Glycine resulted in a conformational and minor environmental changes that decrease chaperone-like activity in the presence of divalent ions suggested that His18, His119 and His101 were a crucial binding site for Cu (II) and Zn (II), respectively. All results together suggest that His18, His119 and His101 coordinate each other for the binding site of Cu (II) and Zn (II) in terms of improving the chaperone-like activity and stability of crystallin/metal ion complex, which further confirmed the proposed binding site based on the theoretical calculation.

1.  Introduction……………………………………………………………………………………………………………6 
1.1 Distribution of α-crystallin…………………………………………………………………6  
1.2 Physical–chemical properties of α-crystallin………………………7
1.3 Chaperone-like function of α-crystallin……………………………………10
1.4 The function of α-crystallin in the lens or in other parts of the visual system…………………………………………………………………………………12
1.5 Diseases involving α-crystallin…………………………………………………………15
1.6 Motive/purpose of the research…………………………………………………………………18                                                   
2.  Materials and Methods……………………………………………………………………………………21
2.1 Plasmid constructions……………………………………………………………………………………21
2.2 Expression and purification of wild type and mutant αB-crystalline…………………………………………………………………………………………………………………………22
2.3 Chaperone-like activity………………………………………………………………………………22
2.4 Fluorescence studies………………………………………………………………………………………23
2.5 CD spectral studies…………………………………………………………………………………………23
2.6 Size-exclusion chromatography………………………………………………………………24
2.7 Statistical analysis………………………………………………………………………………………25
3.  Results and Discussions………………………………………………………………………………26
3.1 Effects of divalent metal ions on the chaperone activity and structure of rat lens H18G mutant αB-crystallin……………………………………………………………………………………………………………………………26
3.1.1 Chaperone-like activity of αB-crystallin and H18G mutant in the presence or absence of Cu (II), Zn (II) and Mg (II) ions………………………………………………………………………………………………………………………26
3.1.2 ANS fluorescence of αB-crystallin and H18G mutant in the presence or absence of Cu (II), Zn (II) and Mg (II) ions……………………………………………………………………………………………………………………………………………27
3.1.3 Secondary and tertiary structure of αB-crystallin and H18G mutant with or without the presence of divalent metal ions……………………………………………………………………………………………………………………………………………29
3.1.4 Effects on aggregation of wild type and H18G mutant αB-crystallin with or without the presence of divalent metal ions……………………………………………………………………………………………………………………………………………31
3.2 Effects of divalent metal ions on the chaperone activity and structure of rat lens H119G mutant αB-crystallin……………………………………………………………………………………………………………………………34
3.2.1 Chaperone-like activity of αB-crystallin and H119G mutant in the presence or absence of Cu (II), Zn (II) and Mg (II) ions………………………………………………………………………………………………………………………34
3.2.2 ANS fluorescence of αB-crystallin and H119G mutant in the presence or absence of Cu (II), Zn (II) and Mg (II) ions……………………………………………………………………………………………………………………………………………35
3.2.3 Secondary and tertiary structure of αB-crystallin and H119G mutant with or without the presence of divalent metal ions……………………………………………………………………………………………………………………………………………37
3.3 Effects of divalent metal ions on the chaperone activity and structure of rat lens H101G mutant αB-crystallin……………………………………………………………………………………………………………………………41
3.3.1 Chaperone-like activity of αB-crystallin and H101G mutant in the presence or absence of Cu (II), Zn (II) and Mg (II) ions………………………………………………………………………………………………………………………41
3.3.2 ANS Fluorescence of αB-Crystallin and H101G mutant in the presence and absence of Cu (II), Zn (II) and Mg (II) ions……………………………………………………………………………………………………………………………………………42
3.3.3 Secondary and tertiary structure of αB-crystallin and H101G mutant with or without the presence of divalent metal ions……………………………………………………………………………………………………………………………………………43
4. Conclusions ………………………………………………………………………………………………………………46
4.1 Effects of divalent metal ions on the molecular chaperone activity and structure of rat lens H18G mutant αB-crystallin……………………………………………………………………………………………………………………………46
4.2 Effects of divalent metal ions on the molecular chaperone activity and structure of rat lens H119G mutant αB-crystallin……………………………………………………………………………………………………………………48
4.3 Effects of divalent metal ions on the molecular chaperone activity and structure of rat lens H101G mutant αB-crystallin……………………………………………………………………………………………………………………51
4.4 The comparisons between chaperone-like activity and αB-crystallin structure of wild type and mutant H18G, H101G, and H119G with or without the presence of metal ions……………53
5. References……………………………………………………………………………………………………………………57
   
Figures and Tables………………………………………………………………………………………………………73
Figure 1. The Plasmid constructions cloned to the pET-19b expression vector…………………………………………………………………………………………………………73
Figure 2. The plasmid constructs of the mutagenesis study of rat lens αB-crystallin at H18G, H101G, and H119G were confirmed by DNA sequencing………………………………………………………………………………74
Figure 3. The wild type and mutant type αB-crystallin containing fractions were identified by SDS-PAGE………………………75
Figure 4. Effect of divalent metal ions (1 mM) on the DTT-induced aggregation of insulin in the presence of αB-crystallin and H18G mutant…………………………………………………………………………………76
Figure 5. The ANS fluorescence spectra of B-crystallin and H18G mutant in the absence and presence of bivalent metal ions (1 mM) with 20μM ANS……………………………………………………………………77
Figure 6. The Far- and Near-UV CD spectra of αB-crystallin and H18G mutant in the presence and absence of divalent metal ions……………………………………………………………………………………………………………………………78
Figure 7. Size-exclusion chromatography profile of αB-crystallin and H18G mutant in the absence and presence of bivalent metal ions……………………………………………………………………………………………………79
Figure 8. Effect of divalent metal ions (1 mM) on the DTT-induced aggregation of insulin in the presence of αB-crystallin and H119G mutant………………………………………………………………………………80
Figure 9. The ANS fluorescence spectra of αB-crystallin and H119G mutant in the absence and presence of bivalent metal ions (1 mM) with 20μM ANS……………………………………………………………………………………81
Figure 10. The Far- and Near-UV CD spectra of αB-crystallin and H119G mutant in the presence and absence of divalent metal ions……………………………………………………………………………………………………………………………82
Figure 11. Effect of divalent metal ions (1 mM) on the DTT-induced aggregation of insulin in the presence of αB-crystallin and H101G mutant………………………………………………………………………………83
Figure 12. The ANS fluorescence spectra of αB-crystallin and H101G mutant in the absence and presence of bivalent metal ions (1 mM) with 20 μM ANS…………………………………………………………………84
Figure 13. The Far- and Near-UV CD spectra of αB-crystallin and H101G mutant in the presence and absence of divalent metal ions……………………………………………………………………………………………………………………………85
Figure 14. Comparison in the percentage of protection ability between wild type and mutants in the presence and absence of metal ions.……………………………………………………………………………………………86
Figure 15. Comparison in the hydrophobicity of wild type and mutants with or without the presence of metal ions………87
Table 1. The content of secondary structural elements for αB-crystallin and H18G mutant in the presence and absence of bivalent metal ions at room temperature based on the far-UV CD spectra……………………………………………………………………………………………………………………88
Table 2. The content of secondary structural elements for αB-crystallin and H119G mutant in the presence and absence of bivalent metal ions at room temperature based on the far-UV CD spectra……………………………………………………………………………………………………………………89
Table 3. The content of secondary structural elements for αB-crystallin and H101G mutant in the presence and absence of bivalent metal ions at room temperature based on the far-UV CD spectra……………………………………………………………………………………………………………………90
Table 4. The content of secondary structural elements for αB-crystallin and mutants in the presence and absence of bivalent metal ions at room temperature based on the far-UV CD spectra……………………………………………………………………………………………………………………………91

Appendix 
Appendix 1. The view of: (a) residues His18, His101, His119, Glu99 and Lys121 in coordination with Cu (II); (b) stereoplot of αB-crystallin, showing the secondary structure in magenta…………………………………………………………………………………………………92
Appendix 2. Simplified model for the coordination site of zinc with the specified atomic partial charges……………………………93
 

                                    

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