Lampi Kirsten J, Kim Yung H, Bächinger Hans Peter, Boswell Bruce A, Lindner Robyn A, Carver John A, Shearer Thomas R, David Larry L, Kapfer Deborah M
Department of Oral Molecular Biology, Oregon Health and Science University, Portland, OR, USA.
Mol Vis. 2002 Sep 25;8:359-66.
To determine how deamidation and partial loss of the N- and C-terminal extensions alter the heat stability of betaB1-crystallin.
Human lens betaB1, a deamidated betaB1, Q204E, and alphaA-crystallins were expressed. Truncated betaB1 was generated by proteolytic removal of part of its terminal extensions. The aggregation and precipitation of these proteins due to heating was monitored by circular dichroism and light scattering. The effect of heat on the stability of both monomers and oligomers was investigated. The flexibility of the extensions in wild type and deamidated betaB1 was assessed by 1H NMR spectroscopy.
With heat, deamidated betaB1 precipitated more readily than wild type betaB1. Similar effects were obtained for either monomers or oligomers. Flexibility of the N-terminal extension in deamidated betaB1 was significantly reduced compared to the wild type protein. Truncation of the extensions further increased the rate of heat-induced precipitation of deamidated betaB1. The presence of the molecular chaperone, alphaA-crystallin, prevented precipitation of modified betaB1s. More alphaA was needed to chaperone the truncated and deamidated betaB1 than deamidated betaB1 or truncated betaB1.
Deamidation and truncation of betaB1 led to destabilization of the protein and decreased stability to heat. Decreased stability of lens crystallins may contribute to their insolubilization and cataract formation.
确定N端和C端延伸部分的脱酰胺作用及部分缺失如何改变βB1-晶体蛋白的热稳定性。
表达人晶状体βB1、脱酰胺化的βB1（Q204E）和αA-晶体蛋白。通过蛋白水解去除其部分末端延伸部分来生成截短的βB1。通过圆二色性和光散射监测这些蛋白质因加热而发生的聚集和沉淀。研究了热对单体和寡聚体稳定性的影响。通过1H NMR光谱评估野生型和脱酰胺化βB1中延伸部分的柔韧性。
加热时，脱酰胺化的βB1比野生型βB1更容易沉淀。单体或寡聚体均获得类似效果。与野生型蛋白相比，脱酰胺化βB1中N端延伸部分的柔韧性显著降低。延伸部分的截短进一步增加了脱酰胺化βB1热诱导沉淀的速率。分子伴侣αA-晶体蛋白的存在可防止修饰的βB1沉淀。与脱酰胺化βB1或截短的βB1相比，截短且脱酰胺化的βB1需要更多的αA来进行伴侣作用。
βB1的脱酰胺化和截短导致蛋白质不稳定并降低热稳定性。晶状体晶体蛋白稳定性的降低可能导致其不溶性增加和白内障形成。
