Linear free-energy model description of the conformational stability of uracil-DNA glycosylase inhibitor

Abstract

The equilibrium unfolding of uracil DNA glycosylase inhibitor (Ugi), a small acidic protein of molecular mass 9474 Da, has been studied by a combination of thermal-induced and guanidine hydrochloride (GdnCl)-induced denaturation. The analysis of the denaturation data provides a measure of the changes in conformational free energy, enthalpy, entropy and heat capacity δ C_p that accompany the equilibrium unfolding of Ugi over a wide range of temperature and GdnCl concentration. The unfolding of Ugi is a simple two-state, reversible process. The protein undergoes both low-temperature and high-temperature unfolding even in the absence of GdnCl but more so in the presence of denaturant. The data are consistent with the linear free-energy model and with a temperature independent δ C_p over the large temperature range of unfolding. The small δ C_p (6.52 kJ·mol^≄1·K⁻¹) for the unfolding of Ugi, is perhaps a reflection of a relatively small, buried hydrophobic core in the folded form of this small monomeric protein. Despite a relatively low value of δ G_(H2O), 7.40 kJ·mol⁻¹ at pH 8.3, Ugi displays considerable stability with the temperature of maximum stability being 301.6 K.