保守色氨酸对维持βB2晶状体蛋白稳定性的重要性以及光保护机制研究

βB2-Crystallin is one of the major protein constituents of the human lens, its long-term solubility and stability are critical for lens transparency and refraction. βB2-crystallin has several highly conserved buried tryptophans, mutations of these tryptophans will lead to congenital cataract. However, the pathopoiesis mechanism remains elusive. Ultraviolet (UV) radiation is one of the risk factors for the age-related cataract. In situ, these Trps will be absorbing ambient UV radiation that reaches the lens. Trp fluorescence is efficiently quenched in native HγD-Crystallin by intradomain Resonance Energy Transfer and fast electron transfer to the amide backbone. Here we constuct site-directed mutants of βB2-crystallin, and product recombinant proteins by overexpressing them in E.coli. Then the effect of mutations on βB2-crystallin structure and stability is investigated by biophysical methods. Meanwhile, we take quantitative measurements of the fluorescence emission spectra and the quantum yields, as well as the lifetime of excited-state Trps by time-resolved fluorescence. Moreover, hybrid quantum mechanical-molecular mechanical (QM-MM) simulations are carried out to predict the quenching mechanism. During our research, through elucidating role of conserved tryptophans in βB2-crystallin’s stability and their photoprotective mechanism by both experiments and computation, we can get a better understanding of the relationship between protein alteration and cataractogenesis, as well as creating a foundation which will help us in further prevention and cure of catatact.

βB2晶状体蛋白是人眼晶状体中重要的结构蛋白，与维持晶状体的透明性与屈光性密切相关。该蛋白拥有数个高度保守的氨基酸位点，发生突变会导致先天性白内障，但具体的致病机制仍未知。紫外线辐射造成的蛋白光损伤是年龄相关性白内障的致病因素之一，到达晶状体的紫外线主要被色氨酸吸收，已知晶状体蛋白的保守色氨酸具有特殊的荧光淬灭过程来实现蛋白的光保护，而类似机制是否适用于βB2晶状体蛋白尚不明确。本课题拟将βB2晶状体蛋白的保守色氨酸以定点突变的方法分别进行替换，采用原核表达获取重组蛋白，研究突变前后蛋白结构和稳定性方面的变化，同时通过色氨酸的相关荧光参数明确是否存在共振能量转移与荧光淬灭，并采用QM-MM方法模拟荧光淬灭过程。本课题旨在较完整地揭示βB2晶状体蛋白的保守色氨酸对维持蛋白稳定性作用以及色氨酸参与蛋白光保护的具体机制，从而更加明确白内障的发生机理，为白内障的防治工作提供理论依据。

βB2晶状体蛋白是人眼晶状体中重要的结构蛋白，与维持晶状体的透明性与屈光性密切相 关。该蛋白拥有数个高度保守的氨基酸位点，发生突变会导致先天性白内障，但具体的致病机 制仍未知。紫外线辐射造成的蛋白光损伤是年龄相关性白内障的致病因素之一，到达晶状体的 紫外线主要被色氨酸吸收，已知晶状体蛋白的保守色氨酸具有特殊的荧光淬灭过程来实现蛋白的光保护，而类似机制是否适用于βB2晶状体蛋白尚不明确。本课题首次在一个白内障家系中发现βB2晶状体蛋白的新突变W151R将βB2晶状体蛋白的保守色氨酸以定点突变的方法分别进行替换，采用原核表达获取重组蛋白，通过生物物理，细胞学及分子动力学模拟研究突变前后蛋白结构和稳定性方面的变化。本课题，并重点对针对W59和W151这两个位于βB2 晶状体蛋白结构域疏水核心的保守色氨酸位点进行了研究。课题发现，晶状体蛋白W59和W151位氨基酸突变后，蛋白结构稳定及热稳定性下降，疏水性暴露增多最终导致聚集形成并诱导凋亡。羊毛甾醇作为近年来发现的潜在逆转白内障的小分子，可以有效缓解突变带来的影响。该课题，从分子层面探讨了白内障的形成机制，探究了羊毛甾醇在预防蛋白质聚集过程中的作用，为白内障的防治提供了一定理论依据。