The effect of copper/zinc metal ion replacement on the folding free energy of wild type (w.t.) and disulfide bridge depleted (C3A/C26A) azurin has been investigated by differential scanning calorimetry (DSC) and fluorescence techniques. The denaturation expts. have shown that, in both cases, the thermal transitions of the zinc deriv. of azurins can be depicted in terms of the classical Lumry-Eyring model, N.tautm.U®F, thus resembling the unfolding path of the two copper proteins. The thermally induced transition of Zn azurin, monitored by fluorescence occurs at lower temp. than the DSC scans indicating that a local conformational rearrangement of the Trp microenvironment, takes place before protein denaturation. For Zn C3A/C26A azurin, the two techniques reveal the same transition temp. Comparison of the thermodn. data shows that the presence of Zn in the active site stabilizes the three-dimensional structure of azurin only when the disulfide bridge is present. Compared to the copper form of the protein, the unfolding temp. of Zn azurin has increased by 4°, while the unfolding free energy, DG, is 31 kJ/mol higher. Both enthalpic and entropic factors contribute to the obsd. DG increase. However, the copper/zinc replacement has no effect on the unfolding free energy of C3A/C26A azurin. Taking Cu azurin w.t. as the ref. state, for both Cu and Zn C3A/C26A azurin the unfolding free energy is decreased by about 28 kJ/mol, indicating that metal substitution is not able to compensate the destabilizing effect induced by the disulfide bridge depletion. It is noteworthy that the thermal denaturation of the Zn deriv., which thermodynamically is the most stable form of azurin, is also characterized by the highest value of the activation energy, Ea, as derived from the kinetic stability anal.
The effect of copper/zinc replacement on the folding free energy of wild type and Cys3Ala/Cys26Ala azurin.
Milardi D;
2003
Abstract
The effect of copper/zinc metal ion replacement on the folding free energy of wild type (w.t.) and disulfide bridge depleted (C3A/C26A) azurin has been investigated by differential scanning calorimetry (DSC) and fluorescence techniques. The denaturation expts. have shown that, in both cases, the thermal transitions of the zinc deriv. of azurins can be depicted in terms of the classical Lumry-Eyring model, N.tautm.U®F, thus resembling the unfolding path of the two copper proteins. The thermally induced transition of Zn azurin, monitored by fluorescence occurs at lower temp. than the DSC scans indicating that a local conformational rearrangement of the Trp microenvironment, takes place before protein denaturation. For Zn C3A/C26A azurin, the two techniques reveal the same transition temp. Comparison of the thermodn. data shows that the presence of Zn in the active site stabilizes the three-dimensional structure of azurin only when the disulfide bridge is present. Compared to the copper form of the protein, the unfolding temp. of Zn azurin has increased by 4°, while the unfolding free energy, DG, is 31 kJ/mol higher. Both enthalpic and entropic factors contribute to the obsd. DG increase. However, the copper/zinc replacement has no effect on the unfolding free energy of C3A/C26A azurin. Taking Cu azurin w.t. as the ref. state, for both Cu and Zn C3A/C26A azurin the unfolding free energy is decreased by about 28 kJ/mol, indicating that metal substitution is not able to compensate the destabilizing effect induced by the disulfide bridge depletion. It is noteworthy that the thermal denaturation of the Zn deriv., which thermodynamically is the most stable form of azurin, is also characterized by the highest value of the activation energy, Ea, as derived from the kinetic stability anal.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
