Equilibrium unfolding studies of horse muscle acylphosphatase.

The stability and equilibrium unfolding behaviour of horse muscle acylphosphatase have been studied by denaturing the protein under various conditions of temperature, pH, and urea concentration. Far-ultraviolet circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy indicate that this small monomeric protein unfolds reversibly and cooperatively. Thermodynamic parameters, the Gibbs free energy delta G and enthalpy delta H of unfolding, have been estimated for denaturation of the protein from NMR and CD data as 19 kJ mol-1 and 350 kJ mol-1, respectively. CD and 1H-NMR results suggest the presence of very little persistent residual structure in the denatured states studied under these different conditions. Furthermore, photo-chemically induced dynamic nuclear polarisation experiments show that in the denatured states aromatic residues are freely accessible to a flavin dye probe.