Cataract is the major reason for human blindness worldwide. α-Crystallin, as a key chaperone of eye lenses, keeps the lenticular tissues in its transparent state over time. In this study, cataract-causing familial mutations, P20R and A171T, were introduced in CRYАB gene. After successful expression in Escherichia coli and subsequent purification, the recombinant proteins were subjected to extensive structural and functional analyses using various spectroscopic techniques, gel electrophoresis, and electron microscopy. The results of fluorescence and Raman assessments suggest important but discreet conformational changes in human αB-Cry upon these cataractogenic mutations. Furthermore, the mutant proteins exhibited significant secondary structural alteration as revealed by FTIR and Raman spectroscopy. An increase in conformational stability was seen in the human αB-Cry bearing these congenital cataractogenic mutations. The oligomeric size distribution and chaperone-like activity of human αB-Cry were significantly altered by these mutations. The P20R mutant protein was observed to loose most of the chaperone-like activity. Finally, these cataractogenic mutant proteins exhibited an increased propensity to form the amyloid fibrils when incubated under environmental stress. Overall, the structural and functional changes in mutated human αB-Cry proteins can shed light on the pathogenic development of congenital cataracts.

中文翻译：

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先天性白内障突变 P20R 和 A171T 与人 αB-晶状体蛋白的淀粉样蛋白生成特征、结构和分子伴侣样活性的重要变化有关

白内障是全世界人类失明的主要原因。α-Crystallin 作为晶状体的关键伴侣，随着时间的推移，使晶状体组织保持透明状态。在这项研究中，导致白内障的家族性突变 P20R 和 A171T 被引入到 CRY-B 基因中。在大肠杆菌中成功表达并随后纯化后，使用各种光谱技术、凝胶电泳和电子显微镜对重组蛋白进行广泛的结构和功能分析。荧光和拉曼评估的结果表明，这些白内障突变导致人类 αB-Cry 发生了重要但谨慎的构象变化。此外，FTIR 和拉曼光谱显示，突变蛋白表现出显着的二级结构改变。在携带这些先天性白内障突变的人类 αB-Cry 中观察到构象稳定性的增加。这些突变显着改变了人 αB-Cry 的寡聚体大小分布和分子伴侣样活性。观察到 P20R 突变蛋白失去了大部分的伴侣蛋白样活性。最后，当在环境压力下孵育时，这些致白内障的突变蛋白表现出形成淀粉样原纤维的倾向增加。总体而言，突变的人类 αB-Cry 蛋白的结构和功能变化可以揭示先天性白内障的致病发展。观察到 P20R 突变蛋白失去了大部分的伴侣蛋白样活性。最后，当在环境压力下孵育时，这些致白内障的突变蛋白表现出形成淀粉样原纤维的倾向增加。总体而言，突变的人类 αB-Cry 蛋白的结构和功能变化可以揭示先天性白内障的致病发展。观察到 P20R 突变蛋白失去了大部分的伴侣蛋白样活性。最后，当在环境压力下孵育时，这些致白内障的突变蛋白表现出形成淀粉样原纤维的倾向增加。总体而言，突变的人类 αB-Cry 蛋白的结构和功能变化可以揭示先天性白内障的致病发展。