The thermal unfolding of azurin in D2O has been investigated by differential scanning calorimetry, optical density measurements, and electron paramagnetic resonance spectroscopy, The study has allowed us to relate the local conformational changes occurring around the active site of azurin with the unfolding of the whole protein as the temperature increases. DSC and OD experiments have shown that the thermal unfolding is, on the whole, irreversible and kinetically controlled. Moreover, by extrapolation of both the experimental heat capacity and the optical density data to infinite heating rate, we have separated the reversible step from the irreversible, kinetically controlled one and calculated the thermodynamic parameters of the time-independent part of the denaturation process. The whole of the results suggest that the unfolding of azurin in D2O follows the same pathway as observed in H2O (La Rosa et al. J. Phys. Chem. 1995, 99, 14864-14870),(1) but it is shifted to a higher temperature. From the comparison of the thermodynamic unfolding parameters obtained in the two solvents, it results that D2O destabilizes the native state of the protein. According to an analysis of the thermodynamic behavior of model compounds in heavy water, this destabilizing effect can be mainly ascribed to the apolar groups of the protein. In addition, the region around the active site is enthalpically less influenced by changing the solvent with respect to the global protein. This behavior has been ascribed to the different solvent-azurin interactions in heavy and in light water. Finally, EPR results show that during the thermal unfolding, the active-site geometry changes from trigonal bipyramidal to square planar. Such a conformational change is not influenced by solvent isotopic effects.
|Titolo:||Solvent isotope effects on azurin thermal unfolding|
|Autori interni:||LA ROSA, Carmelo|
|Data di pubblicazione:||1998|
|Rivista:||JOURNAL OF PHYSICAL CHEMISTRY. B, CONDENSED MATTER, MATERIALS, SURFACES, INTERFACES & BIOPHYSICAL|
|Appare nelle tipologie:||1.1 Articolo in rivista|