Bateman O A, Sarra R, van Genesen S T, Kappé G, Lubsen N H, Slingsby C
Department of Crystallography, Birkbeck College, London, UK.
Exp Eye Res. 2003 Oct;77(4):409-22. doi: 10.1016/s0014-4835(03)00173-8.
Crystallins are bulk structural proteins of the eye lens that have to last a life time. They gradually become modified with age, denature and form light scattering centres. High thermodynamic and kinetic stability of the crystallins enables them to resist unfolding and delay cataract. Here we have made recombinant human betaA1-, betaA3-, and betaA4-crystallins. The betaA3-crystallin formed higher oligomers that lead to precipitation at ambient temperature. Heat-induced precipitation of betaA3-crystallin was compared with human and calf betaB2-crystallins, showing that the human proteins start to precipitate above 50 degrees C while the calf betaB2-crystallin stays in solution even when unfolded. The stabilities of these human acidic beta-crystallin homo-oligomers have been estimated by measuring their unfolding in urea at neutral pH. BetaA3/1/betaB1 and betaA4/betaB1-crystallin hetero-oligomers have been prepared from homo-oligomers by subunit exchange. The resolution of the methodology used was insufficient to detect a stabilization of the betaA4-crystallin subunit in the hetero-oligomer, the betaA1-crystallin subunit was clearly stabilized by its interaction with betaB1-crystallin. Circular dichroism and fluorescence spectroscopies show that homo-dimer surface tryptophans become buried in the betaA3/1/betaB1-crystallin hetero-dimer concomitant with changes in polypeptide chain conformation.
晶状体蛋白是眼晶状体的主要结构蛋白，必须维持一生。随着年龄增长，它们会逐渐发生修饰、变性并形成光散射中心。晶状体蛋白的高热力学和动力学稳定性使其能够抵抗解折叠并延缓白内障的发生。在此，我们制备了重组人βA1-、βA3-和βA4-晶状体蛋白。βA3-晶状体蛋白形成了更高的寡聚体，导致在室温下沉淀。将βA3-晶状体蛋白的热诱导沉淀与人和小牛的βB2-晶状体蛋白进行了比较，结果表明人源蛋白在50摄氏度以上开始沉淀，而小牛βB2-晶状体蛋白即使在解折叠时仍能保持在溶液中。通过在中性pH条件下测量这些人酸性β-晶状体蛋白同型寡聚体在尿素中的解折叠情况，估算了它们的稳定性。通过亚基交换从同型寡聚体制备了βA3/1/βB1和βA4/βB1-晶状体蛋白异型寡聚体。所用方法的分辨率不足以检测到异型寡聚体中βA4-晶状体蛋白亚基的稳定性增强，而βA1-晶状体蛋白亚基通过与βB1-晶状体蛋白的相互作用明显得到了稳定。圆二色性和荧光光谱表明，同型二聚体表面的色氨酸在βA3/1/βB1-晶状体蛋白异型二聚体中被掩埋，同时伴随着多肽链构象的变化。
