Guzzi R, Sportelli L, La Rosa C, Milardi D, Grasso D, Verbeet M P, Canters G W
Dipartimento di Fisica e Unità INFM, Laboratorio di Biofisica Molecolare, Università della Calabria, 87030 Rende (CS), Italy.
Biophys J. 1999 Aug;77(2):1052-63. doi: 10.1016/S0006-3495(99)76955-9.
The disulfide bond connecting Cys-3 and Cys-26 in wild type azurin has been removed to study the contribution of the -SS- bond to the high thermal resistance previously registered for this protein (. J. Phys. Chem. 99:14864-14870). Site-directed mutagenesis was used to replace both cysteines for alanines. The characterization of the Cys-3Ala/Cys-26Ala azurin mutant has been carried out by means of electron paramagnetic resonance spectroscopy at 77 K, UV-VIS optical absorption, fluorescence emission and circular dichroism at room temperature. The results show that the spectral features of the Cys-3Ala/Cys-26Ala azurin resemble those of the wild type azurin, indicating that the double mutation does not affect either the formation of the protein's overall structure or the assembly of the metal-binding site. The thermal unfolding of the Cys-3Ala/Cys-26Ala azurin has been followed by differential scanning calorimetry, optical absorption variation at lambda(max) = 625 nm, and fluorescence emission using 295 nm as excitation wavelength. The analysis of the data shows that the thermal transition from the native to the denaturated state of the modified azurin follows the same multistep unfolding pathway as observed in wild type azurin. However, the removal of the disulfide bridge results in a dramatic reduction of the thermodynamic stability of the protein. In fact, the transition temperatures registered by the different techniques are down-shifted by about 20 degrees C with respect to wild type azurin. Moreover, the Gibbs free energy value is about half of that found for the native azurin. These results suggest that the disulfide bridge is a structural element that significantly contributes to the high stability of wild type azurin.
野生型天青蛋白中连接半胱氨酸-3和半胱氨酸-26的二硫键已被去除，以研究-S-S-键对该蛋白先前记录的高热稳定性的贡献（《物理化学杂志》99:14864 - 14870）。采用定点诱变将两个半胱氨酸都替换为丙氨酸。通过在77 K下的电子顺磁共振光谱、室温下的紫外-可见吸收光谱、荧光发射光谱和圆二色光谱对半胱氨酸-3丙氨酸/半胱氨酸-26丙氨酸天青蛋白突变体进行了表征。结果表明，半胱氨酸-3丙氨酸/半胱氨酸-26丙氨酸天青蛋白的光谱特征与野生型天青蛋白相似，表明双重突变既不影响蛋白质整体结构的形成，也不影响金属结合位点的组装。采用差示扫描量热法、在λ(max)=625 nm处的光吸收变化以及使用295 nm作为激发波长的荧光发射来跟踪半胱氨酸-3丙氨酸/半胱氨酸-26丙氨酸天青蛋白的热解折叠过程。数据分析表明，修饰后的天青蛋白从天然态到变性态的热转变遵循与野生型天青蛋白中观察到的相同的多步解折叠途径。然而，二硫键的去除导致蛋白质的热力学稳定性显著降低。事实上，不同技术记录的转变温度相对于野生型天青蛋白下移了约20℃。此外，吉布斯自由能值约为天然天青蛋白的一半。这些结果表明，二硫键是一个对野生型天青蛋白的高稳定性有显著贡献的结构元件。
