单点氨基酸突变调控白内障相关人γC晶状体蛋白结构的热稳定性

doi:10.16352/j.issn.1001-6325.2025.11.1415

基础医学与临床 ›› 2025, Vol. 45 ›› Issue (11): 1415-1419.doi: 10.16352/j.issn.1001-6325.2025.11.1415

单点氨基酸突变调控白内障相关人γC晶状体蛋白结构的热稳定性

刘明巍¹, 陈明锐², 王晨轩², 张文博^2*

1.信阳职业技术学院, 河南信阳 465400;
2.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

收稿日期:2024-11-21 修回日期:2025-02-20 出版日期:2025-11-05 发布日期:2025-10-24
通讯作者: ^*zwb@ibms.pumc.edu.cn
基金资助:
国家自然科学基金(32201142);信阳职业技术学院校级科研基金(LXQN231102)

Single-site mutation regulates thermal stability of cataract-related human γC crystallin protein structure

LIU Mingwei¹, CHEN Mingrui², WANG Chenxuan², ZHANG Wenbo^2*

1. Xinyang Vocational and Technical College, Xinyang 465400;
2. Department of Biophysics and Structural Biology, State Key Laboratory of Common Mechanism Research for Major Diseases, Institute of Basic Medical Sciences CAMS, School of Basic Medicine PUMC, Beijing 100005, China

Received:2024-11-21 Revised:2025-02-20 Online:2025-11-05 Published:2025-10-24
Contact: ^*zwb@ibms.pumc.edu.cn

摘要/Abstract

摘要： 目的揭示与先天性白内障相关的第129位甘氨酸到半胱氨酸的点突变(G129C)调控人γC晶状体蛋白(HγC)结构稳定性的分子机制。方法体外表达纯化野生型HγC蛋白(HγC-WT)与其G129C突变体HγC-G129C,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规律,对比HγC-WT与HγC-G129C的折叠和聚集结构对温度的依赖关系。结果当温度低于65 ℃,HγC-WT与HγC-G129C的内源荧光质心波长随温度升高而向较长波长偏移,荧光强度降低,蛋白质构象去折叠。当温度高于65 ℃,静态光散射强度随温度升高而显著上升,蛋白质受热聚集。相对于HγC-WT,HγC-G129C表现出更强的聚集趋势。对于HγC-WT与HγC-G129C的受热变性过程,交叉点温度分别是74.5 ℃和55.5 ℃。HγC-WT展示出更高的热稳定性。结论先天性白内障相关的G129C点突变显著降低了γC晶状体蛋白的结构的热稳定性。

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

Abstract: Objective To find the molecular mechanism underlying the effect of congenital cataract related 129^th single-site mutation G129C on the thermal stability of human γC crystallin (HγC) protein structure. Methods HγC-WT and HγC-G129C were expressed and purified in vitro. The changes of intrinsic fluorescence intensity and static light scattering intensity of proteins with temperature were measured, and the temperature dependence of the folding and aggregation structures of HγC-WT and HγC-G129C was compared. Results When temperature was below 65 ℃, the barycentric mean of the intrinsic fluorescence of HγC-WT and HγC-G129C shifted towards a longer wavelength and the fluorescence intensity decreased with the increasing temperature, which was believed to be the evidence of unfolded protein conformation. When the temperature was higher than 65 ℃, the static light scattering intensity increased significantly with the temperature, indicating the protein aggregation upon heating. The wild-type HγC-G129C showed a stronger aggregation potency. During the thermal de-naturation process of HγC-WT and HγC-G129C, the crossing-point temperatures were 74.5 ℃ and 55.5 ℃, respectively. HγC-WT showed higher thermal stability. Conclusions The congenital cataract-associated G129C mutation significantly weakens conformational stability of γC-crystallin.

Key words: crystallin, thermal stability, aggregation, intrinsic fluorescence, static light scattering

中图分类号:

刘明巍, 陈明锐, 王晨轩, 张文博. 单点氨基酸突变调控白内障相关人γC晶状体蛋白结构的热稳定性[J]. 基础医学与临床, 2025, 45(11): 1415-1419.

LIU Mingwei, CHEN Mingrui, WANG Chenxuan, ZHANG Wenbo. Single-site mutation regulates thermal stability of cataract-related human γC crystallin protein structure[J]. Basic & Clinical Medicine, 2025, 45(11): 1415-1419.

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单点氨基酸突变调控白内障相关人γC晶状体蛋白结构的热稳定性

Single-site mutation regulates thermal stability of cataract-related human γC crystallin protein structure

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