THERMODYNAMIC CHARACTERIZATION OF AN EQUILIBRIUM FOLDING INTERMEDIATEOF STAPHYLOCOCCAL NUCLEASE

High-sensitivity differential scanning calorimetry and CD spectroscopy have been used to probe the structural stability and measure the fold ing/unfolding thermodynamics of a Pro(117) --> Gly variant of staphylo coccal nuclease. It is shown that at neutral pH the thermal denaturati on of this protein is well accounted for by a 2-state mechanism and th at the thermally denatured state is a fully hydrated unfolded polypept ide. At pH 3.5, thermal denaturation results in a compact denatured st ate in which most, if not all, of the helical structure is missing and the beta subdomain apparently remains largely intact. At pH 3.0, no t hermal transition is observed and the molecule exists in the compact d enatured state within the 0-100 degrees C temperature interval. At hig h salt concentration and pH 3.5, the thermal unfolding transition exhi bits 2 cooperative peaks in the heat capacity function, the first one corresponding to the transition from the native to the intermediate st ate and the second one to the transition from the intermediate to the unfolded state. As is the case with other proteins, the enthalpy of th e intermediate is higher than that of the unfolded state at low temper atures, indicating that, under those conditions, its stabilization mus t be of an entropic origin. The folding intermediate has been modeled by structural thermodynamic calculations. Structure-based thermodynami c calculations also predict that the most probable intermediate is one in which the beta subdomain is essentially intact and the rest of the molecule unfolded, in agreement with the experimental data. The struc tural features of the equilibrium intermediate are similar to those of a kinetic intermediate previously characterized by hydrogen exchange and NMR spectroscopy.