Stability of p53 Homologs

Most proteins have not evolved for maximal thermal stability. Some are only marginally stable, as for example, the DNA-binding domains of p53 and its homologs, whose kinetic and thermodynamic stabilities are strongly correlated. Here, we applied high-throughput methods using a real-time PCR thermocycler to study the stability of several full-length orthologs and paralogs of the p53 family of transcription factors, which have diverse functions, ranging from tumour suppression to control of developmental processes. From isothermal denaturation fluorimetry and differential scanning fluorimetry, we found that full-length proteins showed the same correlation between kinetic and thermodynamic stability as their isolated DNA-binding domains. The stabilities of the full-length p53 orthologs were marginal and correlated with the temperature of their organism, paralleling the stability of the isolated DNA-binding domains. Additionally, the paralogs p63 and p73 were significantly more stable and long-lived than p53. The short half-life of p53 orthologs and the greater persistence of the paralogs may be biologically relevant.绝大多数蛋白质并非朝着最大化热稳定性的方向演化而来。部分蛋白质的稳定性仅处于勉强维持的水平，例如p53及其同源蛋白的DNA结合结构域（DNA-binding domain），其动力学稳定性（kinetic stability）与热力学稳定性（thermodynamic stability）存在极强的相关性。本研究采用基于实时PCR热循环仪（real-time PCR thermocycler）的高通量实验方法，对p53家族转录因子的多个全长直系同源蛋白（orthologs）与旁系同源蛋白（paralogs）的稳定性展开探究；该家族蛋白功能多样，涵盖肿瘤抑制与发育过程调控等多个领域。通过等温变性荧光法（isothermal denaturation fluorimetry）与差示扫描荧光法（differential scanning fluorimetry）的检测分析，我们发现全长蛋白的动力学稳定性与热力学稳定性之间的相关性，与其单独分离的DNA结合结构域完全一致。全长p53直系同源蛋白的稳定性同样处于边缘水平，且与其宿主生物的生存温度呈显著相关，这一规律与它们单独的DNA结合结构域的稳定性变化趋势一致。此外，旁系同源蛋白p63与p73的稳定性与半衰期均显著高于p53。p53直系同源蛋白的短半衰期与旁系同源蛋白的高持久性，或具有重要的生物学意义。