Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i., Královopolská 135, 612 65 Brno, Czech Republic.
J Am Chem Soc. 2011 May 11;133(18):7190-6. doi: 10.1021/ja201006s. Epub 2011 Apr 14.
We developed an innovative electrochemical method for monitoring conformational transitions in proteins using constant current chronopotentiometric stripping (CPS) with dithiothreitol-modified mercury electrodes. The method was applied to study the effect of oncogenic mutations on the DNA-binding domain of the tumor suppressor p53. The CPS responses of wild-type and mutant p53 showed excellent correlation with structural and stability data and provided additional insights into the differential dynamic behavior of the proteins. Further, we were able to monitor the loss of an essential zinc ion resulting from mutation (R175H) or metal chelation. We envisage that our CPS method can be applied to the analysis of virtually any protein as a sensor for conformational transitions or ligand binding to complement conventional techniques, but with the added benefit that only relatively small amounts of protein are needed and instant results are obtained. This work may lay the foundation for the wide application of electrochemistry in protein science, including proteomics and biomedicine.
我们开发了一种创新的电化学方法，用于使用带有二硫苏糖醇修饰的汞电极的恒电流计时电位溶出（CPS）监测蛋白质构象转变。该方法应用于研究致癌突变对肿瘤抑制因子 p53 的 DNA 结合域的影响。野生型和突变型 p53 的 CPS 响应与结构和稳定性数据具有极好的相关性，并提供了对蛋白质差异动力学行为的深入了解。此外，我们能够监测由于突变（R175H）或金属螯合导致的必需锌离子的丢失。我们设想我们的 CPS 方法可以应用于几乎任何蛋白质的分析，作为构象转变或配体结合的传感器，以补充传统技术，但具有仅需要相对少量蛋白质和即时获得结果的额外优点。这项工作可能为电化学在蛋白质科学中的广泛应用奠定基础，包括蛋白质组学和生物医学。
