白内障相关人γD晶状体蛋白的热诱导变性
Heat-induced denaturation of cataract-related human γ D-crystallin
周鑫 1李珍艳 1李淑媛 1张文博 1王晨轩1
作者信息
- 1. 中国医学科学院基础医学研究所 北京协和医学院基础学院 生物物理及结构生物学系重大疾病共性机制研究全国重点实验室,北京 100005
- 折叠
摘要
目的 揭示人γD晶状体蛋白(HGD)与先天性白内障相关突变体蛋白(HGD P23T)的热诱导变性效应,对比野生型与突变体蛋白质在加热过程中结构变化的差异性.方法 体外表达纯化HGD与HGD P23T,测定蛋白质内源性荧光强度和静态光散射光强随温度的变化,揭示HGD与HGD P23T依赖于温度的折叠与聚集结构变化规律.结果 温度低于 70℃时,HGD与HGD P23T的内源荧光质心波长随温度升高向长波长移动,荧光强度降低,蛋白质构象去折叠.HGD P23T比HGD的构象稳定性差.温度高于 70℃时,静态光散射强度随温度显著上升,蛋白质受热聚集.相对于野生型,HGD P23T表现出更强的聚集趋势.结论 加热破坏γD晶状体蛋白的折叠构象,诱导去折叠态蛋白质发生聚集.疾病相关P23T突变显著降低了γD晶状体蛋白的构象稳定性.
Abstract
Objective To reveal the thermally induced denaturation of wild-type human γ D-crystallin(HGD)and congenital cataract-related mutant(HGD P23T),and compare the differences in the structural changes between wild-type and mutants during a heating process.Methods HGD and HGD P23T were expressed and purified.The temperature-dependent intrinsic fluorescence intensity and static light scattering intensity of the protein samples were measured to reveal the temperature-dependent folding and aggregation structural changes of HGD and HGD P23T.Results When the temperature was below 70℃,the barycentric mean of the intrinsic fluorescence of HGD and HGD P23T shifted towards a longer wavelength with increasing temperature and the fluorescence intensity de-creased indicating the unfolded protein conformations.The conformational stability of HGD P23T was weaker than that of HGD.When temperature was higher than 70℃,the static light scattering intensity increased significantly with temperature,indicating protein aggregation upon heating.Relative to the wild-type,HGD P23T showed a stronger aggregation potency.Conclusions Heating disrupts the folding conformation of Γd-crystallin,induces the unfolded protein to aggregate.The disease-associated P23T mutation significantly reduces the conformational stability of Γd-crystallin.
关键词
晶状体蛋白质/热稳定性/聚集/内源性荧光/静态光散射Key words
crystallin/thermal stability/aggregation/intrinsic fluorescence/static light scattering引用本文复制引用
出版年
2025
国家科技期刊平台
NSTL
万方数据