Age‐related lens cataract is the major cause of blindness worldwide. The mechanisms whereby crystallins, the predominant lens proteins, assemble into large aggregates that scatter light within the lens, and cause cataract, are poorly understood. Due to the lack of protein turnover in the lens, crystallins are long‐lived. A major crystallin, γS, is heavily modified by deamidation, in particular at surface‐exposed N14, N76, and N143 to introduce negative charges. In this present study, deamidated γS was mimicked by mutation with aspartate at these sites and the effect on biophysical properties of γS was assessed via dynamic light scattering, chemical and thermal denaturation, hydrogen‐deuterium exchange, and susceptibility to disulfide cross‐linking. Compared with wild type γS, a small population of each deamidated mutant aggregated rapidly into large, light‐scattering species that contributed significantly to the total scattering. Under partially denaturing conditions in guanidine hydrochloride or elevated temperature, deamidation led to more rapid unfolding and aggregation and increased susceptibility to oxidation. The triple mutant was further destabilized, suggesting that the effects of deamidation were cumulative. Molecular dynamics simulations predicted that deamidation augments the conformational dynamics of γS. We suggest that these perturbations disrupt the native disulfide arrangement of γS and promote the formation of disulfide‐linked aggregates. The lens‐specific chaperone αA‐crystallin was poor at preventing the aggregation of the triple mutant. It is concluded that surface deamidations cause minimal structural disruption individually, but cumulatively they progressively destabilize γS‐crystallin leading to unfolding and aggregation, as occurs in aged and cataractous lenses.

中文翻译：

---

主要晶状体蛋白 γS-晶状体蛋白的累积脱酰胺作用会增加其在展开和氧化过程中的聚集。

年龄相关性晶状体白内障是全世界失明的主要原因。人们对晶状体蛋白（主要的晶状体蛋白）聚集成大的聚集体从而在晶状体内散射光并导致白内障的机制知之甚少。由于晶状体中缺乏蛋白质周转，晶状体蛋白的寿命很长。主要的晶状体蛋白 γS 通过脱酰胺作用进行了大量修饰，特别是在表面暴露的 N14、N76 和 N143 处，以引入负电荷。在本研究中，通过在这些位点用天冬氨酸突变来模拟脱酰胺的 γS，并通过动态光散射、化学和热变性、氢-氘交换以及对二硫化物交联的敏感性来评估对 γS 生物物理特性的影响。与野生型γS相比，每个脱酰胺突变体的一小部分迅速聚集成大的光散射物种，对总散射有显着贡献。在盐酸胍或高温的部分变性条件下，脱酰胺作用导致更快速的展开和聚集，并增加了对氧化的敏感性。三重突变体进一步不稳定，表明脱酰胺作用是累积的。分子动力学模拟预测脱酰胺增强了 γS 的构象动力学。我们认为这些扰动破坏了 γS 的天然二硫化物排列并促进了二硫化物连接的聚集体的形成。晶状体特异性伴侣αA-晶状体蛋白在防止三重突变体聚集方面表现不佳。