Thermodynamic stability of ribonuclease A in alkylurea solutions and preferential solvation changes accompanying its thermal denaturation: A calorimetric and spectroscopic study

The effect of methylurea, N,N'-dimethylurea, ethylurea, and butylurea as we ll as guanidine hydrochloride (GuHCl), urea and pH on the thermal stability , structural properties, and preferential solvation changes accompanying th e thermal unfolding of ribonuclease A (RNase A) has been investigated by di fferential scanning calorimetry (DSC), UV, and circular dichroism (CD) spec troscopy. The results show that the thermal stability of RNase A decreases with increasing concentration of denaturants and the size of the hydrophobi c group substituted on the urea molecule. From CD measurements in the near- and far-UV range, it has been observed that the tertiary structure of RNas e A melts at about 3 degrees C lower temperature than its secondary structu re, which means that the hierarchy in structural building blocks exists for RNase A even at conditions at which according to DSC and UV measurements t he RNase A unfolding can be interpreted in terms of a two-state approximati on. The far-UV CD spectra also show that the final denatured states of RNas e A at high temperatures in the presence of different denaturants including 4.5 M GuHCl are similar to each other but different from the one obtained in 4.5 M GuHCl at 25 degrees C. The concentration dependence of the prefere ntial solvation change Delta Gamma(23), expressed as the number of cosolven t molecules entering or leaving the solvation shell of the protein upon den aturation and calculated from DSC data, shows the same relative denaturatio n efficiency of alkylureas as other methods.