A spectroscopic and calorimetric investigation on the thermal stability ofthe Cys3Ala/Cys26Ala azurin mutant

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 res istance previously registered for this protein (La Rosa et al. 1995. J. Phy s. Chem. 99:14864-14870). Site-directed mutagenesis was used to replace bot h cysteines for alanines. The characterization of the Cys-3Ala/Cys-26Ala az urin mutant has been carried out by means of electron paramagnetic resonanc e spectroscopy at 77 K, UV-VIS optical absorption, fluorescence emission an d circular dichroism at room temperature. The results show that the spectra l 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 for mation of the protein's overall structure or the assembly of the metal-bind ing site. The thermal unfolding of the Cys-3Ala/Cys-26Ala azurin has been f ollowed by differential scanning calorimetry, optical absorption variation at lambda(max) = 625 nm, and fluorescence emission using 295 nm as excitati on wavelength. The analysis of the data shows that the thermal transition f rom 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 temperature s registered by the different techniques are down-shifted by about 20 degre es C with respect to wild type azurin. Moreover, the Gibbs free energy valu e is about half of that found for the native azurin. These results suggest that the disulfide bridge is a structural element that significantly contri butes to the high stability of wild type azurin.