Protein isolation, size-exclusion chromatography, spectroscopy, Uncle stability screens and molecular dynamics simulations were used to assess βA3- and βB1-crystallin thermal stability, structural properties and heteromer formation.
Cells that overexpressed βB1-L116P tended to form aggregates and precipitations under heat-shock stress. Thermal denaturation and time-dependent turbidity experiments showed that thermal stability was significantly impaired. Moreover, protein instability appeared to increase with elevated concentrations detected by the Uncle system. Additionally, βA3 had a relative protective effect on βB1-L116P after heteromers were formed, although βA3 was relatively unstable and was usually protected by basic β-crystallins. Molecular dynamic simulations revealed that L116P mutation altered the hydrophobic residues at the surface around the mutant site, providing solvents more access to the internal and hydrophobic parts of the protein.
Decreased βB1-crystallin thermal stability in the presence of the cataract-related L116P mutation contributes significantly to congenital cataract formation. Moreover, its formation of heteromers with βA3 protects against the low thermal stability of βB1-L116P.
方法:蛋白质分离,尺寸排阻色谱法,光谱学,叔叔稳定性筛选和分子动力学模拟用于评估βA3-和βB1-晶状体蛋白的热稳定性,结构性质和异聚体形成。
结果:过表达βB1-L116P的细胞在热休克应激下倾向于形成聚集体和沉淀。热变性和时间依赖性浊度实验表明,热稳定性显着受损。此外,蛋白质不稳定性似乎随着叔叔系统检测到的浓度升高而增加。此外,βA3在异聚体形成后对βB1-L116P有相对保护作用,尽管βA3相对不稳定,通常被碱性β-晶状体蛋白保护。分子动力学模拟显示,L116P突变改变了突变位点周围表面的疏水残基,为溶剂提供更多接触蛋白质内部和疏水部分的途径。
结论:在存在白内障相关的L116P突变的情况下,βB1-晶状体蛋白热稳定性降低显著促进了先天性白内障的形成。此外,它与βA3形成异聚体可以防止βB1-L116P的低热稳定性。