KINETICS AND THERMODYNAMICS OF THERMAL-DENATURATION IN ACYL CARRIER PROTEIN

The denaturation of Escherichia coli acyl carrier protein (ACP) in buf fers containing both monovalent and divalent cations was followed by v ariable-temperature NMR and differential scanning calorimetry. Both hi gh concentrations of monovalent salts (Na+) and moderate concentration s of divalent salts (Ca2+) raise the denaturation temperature, but cal orimetry indicates that a significant increase in the enthalpy of dena turation is obtained only with the addition of a divalent salt. NMR ex periments in both low ionic strength monovalent buffers and low ionic strength monovalent buffers containing calcium ions show exchange betw een native and denatured forms to be slow on the NMR time scale. Howev er, in high ionic strength monovalent buffers, where the temperature o f denaturation is elevated as it is in the presence of Ca2+, the trans ition is fast on the NMR time scale. These results suggest that monova lent and divalent cations may act to stabilize ACP in different ways. Monovalent ions may nonspecifically balance the intrinsic negative cha rge of this protein in a way that is similar for native, denatured, an d intermediate forms. Divalent cations provide stability by binding to specific sites present only in the native state.