Thermodynamic analysis of the contributions of the copper ion and the disulfide bridge to azurin stability: synergism among multiple depletions

The stabilizing potential of the copper ion and the disulfide bridge in azurin has been explored with the aim of inspecting the ways in which these two factors influence one another. Specifically, whether copper and disulfide contributions to protein stability are additive has been examined. To this aim, the thermal unfolding of a copper-depleted mutant lacking the disulfide bridge between Cys3 and Cys26 (apo C3A/C26A azurin) was studied by differential scanning calorimetry. A comparison of the unfolding parameters of holo and apo C3A/C26A azurin with the apo C3A/C26A protein has shown that the effects of simultaneous copper and disulfide depletion are additive only at two temperatures: T=15 °C and T=67 °C. Within this range the presence of the copper ion and the disulfide bridge has a positive synergistic effect on azurin stability. These findings might have implications for the rational use of the stabilizing potential of copper and disulfides in copper protein engineering.