THERMODYNAMIC STUDIES OF THE CORE HISTONES - IONIC-STRENGTH AND PH-DEPENDENCE OF H2A-H2B DIMER STABILITY

The thermal stability of the core histone dimer H2A-H2B has been studi ed by high-sensitivity differential scanning calorimetry and circular dichroism spectroscopy. The unfolding transition temperature of the 28 kDa H2A-H2B dimer increases as a function of both the ionic strength of the solvent and the total protein concentration. At neutral pH and physiological ionic strength, the thermal denaturation is centered at about 50 degrees C with a corresponding enthalpy change of about 40 kc al/mol of 14 kDa monomer unit and an excess heat capacity of about 1.4 kcal/(K . mol) of 14 kDa monomer unit. The H2A-H2B dimer is stable ma inly between pH 5.5 and 10.5. Below pH 4.0, the system is unfolded at all temperatures. The thermodynamic analysis is performed at low ionic strength where almost complete reversibility is attained, since highe r salt conditions seem to promote aggregation and irreversibility of t he transitions. Analysis of the data shows that at low ionic strength and pH values between 6.5 and 8.5, the H2A-H2B dimer behaves as a high ly cooperative system, melting as a single unit without any detectable intermediates of dissociated, yet folded, H2A and H2B monomers. This is consistent with the observed protein concentration dependence of th e midpoint of the thermal denaturation. The two-state unfolding proces s can be described by the general scheme AB --> 2U, indicating that th e individual H2A and H2B polypeptides are folded, stable entities only when complexed as the H2A-H2B dimer and that the major contribution t o the stabilization of the dimer derives from the coupling between the H2A and H2B interfaces.