Effect of D91G mutation on the structure and function of αA crystallin

• Main Authors: Chen, Kuan-Yu, 陳冠宇
• Other Authors: Lee, Hwei-Jen
• Format: Others
• Language: zh-TW
• Published: 2017
• Online Access: http://ndltd.ncl.edu.tw/handle/a38c6a

碩士 === 國防醫學院 === 生物化學研究所 === 105 === αA crystalline is the eye lenses protein. It belongs to the small heat shock protein with chaperone-like function. αA Crystallin has many different functional domains. The recognition element that recognizes the unfolded substrates , the binding element interacts with the unfolding substrates, and a solubilizing element helps to keep the complexes in solution. This study investigate the effect of D91G point mutations on the structure, thermal stability, molecular size and protective function of αA crystallin. The secondary structure changes were analyzed by CD spectroscopy. When the temperature increased, the secondary structures were changed significantly for wild-type αA crystallin and the D91G mutant αA crystalline. The fluorescence intensity of tryptophan of D91G mutant αA crystallin was 24% higher than that of wild-type protein at room temperature. After incubation, at high temperature the fluorescent of D91G mutant αA crystallin was increased by 10%. No changes in ANS , the fluorescent and λmax of the wild-type and mutant αA crystallin were shown changed under room temperature. After heat treatment fluorescence of D91G mutant αA crystallin was 9% higher than that of wild-type αA crystallin . The λmax of ANS fluorescence are shifted to 480 nm for both proteins. From gel-filtration chromatofraphy analysis, the molecular weight of D91G mutant αA crystallin was 29% higher than that of wild type αA crystallin at room temperature. The D91G mutant αA crystallin formed macromolecule polymer more quickly than wild type at increasing temperature. D91G point mutations reduced the onset of protein aggregation from 95 ° C to 80 ° C, and the maximum protein temperature dropped from 95 ° C to 88 ° C. When wild-type and mutant αA crystallin were mixed with human argininosuccinate lyase(hASL). The wild type αA crystallin in a form of high molecular weight polymer was precipitated at 93° C was re-aggregated and precipitated. The D91G mutant αA crystallin was precipitated at 85 ° C. At the ratio of 1:1 to 1:3, the wild-type αA crystallin showed about 60% of the protection of hASL. When the ratio increase to 1: 4, the protection rate was risen to 70%. The D91G mutant αA crystallin at ratio of 1: 1 to 1: 4 showed the protection at 40~50%. After incubation at room temperature, the protection of wild-type αA crystallin was 62 ± 1.4 % and 48 ± 3% , respectively. As the heating to 70 ° C the protection of Wild-type and D91G mutant αA crystallin were decreased to 29 ± 5.3% and 23 ± 6.3%,respectively. D91G point mutations do not affect the protective function of DTT-induced insulin denaturation. These results indicate that point mutation of D91G will affect the structure, thermal stability and protective function of human argininosuccinate lyase(hASL).