Site-Directed Mutagenesis Studies on the Divalent Metal Ions Binding Site of Rat Lens αB-Crystallin

• Main Authors: Shou-YunLu, 盧守筠
• Other Authors: Fu-Yung Huang
• Format: Others
• Language: en_US
• Published: 2014
• Online Access: http://ndltd.ncl.edu.tw/handle/fwjydk

博士 === 國立成功大學 === 化學系 === 102 === 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.