STRUCTURAL ENERGETICS OF BARSTAR STUDIED BY DIFFERENTIAL SCANNING MICROCALORIMETRY

The energetics of barstar denaturation have been studied by CD and sca nning microcalorimetry in an extended range of pH and salt concentrati on. It was shown that, upon increasing temperature, barstar undergoes a transition to the denatured state that is well approximated by a two -state transition in solutions of high ionic strength. This transition is accompanied by significant heat absorption and an increase in heat capacity. The denaturational heat capacity increment at approximate t o 75 degrees C was found to be 5.6+/-0.3 kJ K-1 mol(-1). In all cases, the value of the measured enthalpy of denaturation was notably lower than those observed for other small globular proteins. In order to exp lain this observation, the relative contributions of hydration and the disruption of internal interactions to the total enthalpy and entropy of unfolding were calculated. The enthalpy and entropy of hydration w ere found to be in good agreement with those calculated for other prot eins, but the enthalpy and entropy of breaking internal interactions w ere found to be among the lowest for all globular proteins that have b een studied. Additionally, the partial specific heat capacity of barst ar in the native state was found to be 0.37+/-0.03 cal K-1 g(-1), whic h is higher than what is observed for most globular proteins and sugge sts significant flexibility in the native state. It is known from stru ctural data that barstar undergoes a conformational change upon bindin g to its natural substrate barnase. Our data, which indicate that bars tar has a loosely packed interior, suggest that high conformational fl exibility of barstar's native structure may play an important role in allowing it to optimize its contacts with barnase upon binding without disrupting favorable, tightly packed internal interactions.