BACKGROUND The eye lens crystallins are highly soluble proteins that are required to last the lifespan of an organism due to low protein turnover in the lens. Crystallin aggregation leads to formation of light-scattering aggregates known as cataract. The G18V mutation of human γS-crystallin (γS-G18V), which is associated with childhood-onset cataract, causes structural changes throughout the N-terminal domain and increases aggregation propensity. The holdase chaperone protein αB-crystallin does not interact with wild-type γS-crystallin, but does bind its G18V variant. The specific molecular determinants of αB-crystallin binding to client proteins is incompletely charcterized. Here, a new variant of γS, γS-G18A, was created to test the limits of αB-crystallin selectivity. METHODS Molecular dynamics simulations were used to investigate the structure and dynamics of γS-G18A. The overall fold of γS-G18A was assessed by circular dichroism (CD) spectroscopy and intrinsic tryptophan fluorescence. Its thermal unfolding temperature and aggregation propensity were characterized by CD and DLS, respectively. Solution-state NMR was used to characterize interactions between αB-crystallin and γS-G18A. RESULTS γS-G18A exhibits minimal structural changes, but has compromised thermal stability relative to γS-WT. The placement of alanine, rather than valine, at this highly conserved glycine position produces minor changes in hydrophobic surface exposure. However, human αB-crystallin does not bind the G18A variant, in contrast to previous observations for γS-G18V, which aggregates at physiological temperature. CONCLUSIONS αB-crystallin is capable of distinguishing between aggregation-prone and function-preserving variants, and recognizing the transient unfolding or minor conformers that lead to aggregation in the disease-related variant. GENERAL SIGNIFICANCE Human αB-crystallin distinguishes between highly similar variants of a structural crystallin, binding the cataract-related γS-G18V variant, but not the function-preserving γS-G18A variant, which is monomeric at physiological temperature.

中文翻译：

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人αB-晶状体蛋白可区分客户蛋白的易聚集变体和功能保留变体。

背景技术晶状体晶状体蛋白是高度可溶性的蛋白质，由于晶状体中的蛋白质周转率低，因此它们需要维持生物体的寿命。晶状体蛋白聚集体导致形成光散射聚集体，称为白内障。人γS-晶状体蛋白（γS-G18V）的G18V突变与儿童期白内障相关，导致整个N末端结构域发生结构变化，并增加了聚集倾向。Holdase伴侣蛋白αB-crystallin不会与野生型γS-crystallin相互作用，但会结合其G18V变异体。与客户蛋白质结合的αB-晶状蛋白的特定分子决定因素是不完整的。在这里，创建了一个新的γS变体γS-G18A，以测试αB-晶状蛋白选择性的极限。方法采用分子动力学模拟方法研究γS-G18A的结构和动力学。γS-G18A的整体折叠通过圆二色性（CD）光谱和固有色氨酸荧光进行评估。其热展开温度和聚集倾向分别用CD和DLS表征。溶液状态NMR用于表征αB-晶状蛋白和γS-G18A之间的相互作用。结果γS-G18A表现出最小的结构变化，但相对于γS-WT却具有较差的热稳定性。在高度保守的甘氨酸位置上放置丙氨酸而不是缬氨酸会在疏水表面暴露中产生微小的变化。但是，与先前在生理温度下聚集的γS-G18V观察到的结果相反，人αB-晶状体蛋白不结合G18A变体。结论αB-晶状体蛋白能够区分易于聚集的变异和保留功能的变异，并识别导致疾病相关变异中聚集的瞬时解折叠或次要构象异构体。一般意义人αB-晶状体蛋白区分结构性晶状体蛋白的高度相似变体，它们结合白内障相关的γS-G18V变体，但不结合功能保留的γS-G18A变体，后者在生理温度下是单体。