A CALORIMETRIC STUDY OF THE THERMAL-STABILITY OF BARSTAR AND ITS INTERACTION WITH BARNASET

The temperature-induced unfolding of single, double, and triple mutant s of barstar, the specific intracellular protein inhibitor of barnase from Bacillus amyloliquefaciens, has been studied by high-sensitivity differential scanning calorimetry. The thermal unfolding of barstar mu tants, where at least one of the two cysteine residues in the molecule had been replaced by alanine, follows a two-state mechanism at neutra l and alkaline pH. The unfolding enthalpy and heat capacity changes ar e slightly lower than those accepted for highly compact, small, globul ar proteins. We have found that at pH 2.5, where barstar seems to be i n a molten globule state, the protein has a heat capacity between that of the native and the unfolded states and shows some tendency for ass ociation. Scanning calorimetry experiments were also extended to the b arstar-barnase complex in the neutral and alkaline pH range. The bindi ng constants obtained from DSC studies are similar to those already ob tained from other (kinetic) studies. The interaction of barstar and ba rnase was also investigated by isothermal calorimetry in various buffe rs within the pH range 6.0-10.0 and a temperature range of 15-35 degre es C. The favorable enthalpy contribution to the binding is about 4 ti mes higher than the entropic one at 25 degrees C. The overall data ana lysis of the combined calorimetric results has led to the thermodynami c characterization of barstar unfolding and the interaction of barstar and barnase over a wide range of temperatures.