THERMODYNAMIC STUDIES OF THE CORE HISTONES - PH AND IONIC-STRENGTH EFFECTS ON THE STABILITY OF THE (H3-H4) (H3-H4)(2) SYSTEM/

The self-associative behavior and the thermal stability of the H3/H4 h istone complex was studied in low-ionic strength conditions by several physicochemical techniques, including differential scanning calorimet ry and circular dichroism spectroscopy, At neutrality, the major molec ular species present in solution is the (H3-H4)(2) tetramer. Its therm odynamic properties cannot be studied directly though, since its therm al denaturation is completely irreversible even at the lowest salt con centrations. However, a complete thermodynamic analysis can be perform ed at low ionic strength and pH 4.5, where the (H3-H4)(2) tetramer is quantitatively dissociated into two H3-H4 dimers and where almost comp lete reversibility of the thermal transitions is attained. The unfoldi ng transition temperature of the 26.5 kDa H3-H4 dimer increases as a f unction of both the ionic strength of the solvent and the total protei n concentration. The thermal denaturation of the H3-H4 dimer is charac terized by the presence of a single calorimetric peak, centered at 58 degrees C, with a corresponding enthalpy change of 25 kcal/mol of a 13 kDa monomer unit and a change in heat capacity upon unfolding of abou t 0.6 kcal/(K mol of 13 kDa monomer unit). The complex between histone s H3 and H4 (tetramer or dimer) is stable between pH 9.5 and 3.0. At p H 1.5, the system is almost completely unfolded at all temperatures. A t low ionic strengths and pH values between 5.0 and 2.5, the H3-H4 dim er behaves as a highly cooperative system, melting as a single unit; i .e. individual H3 and H4 folded monomers are not detectable during the treatment, The two-state mechanism accounting for the unfolding of th e H3-H4 dimer at pH 4.5 is the same as that described for the H2A-H2B dimer at neutrality. Just like for the H2A and H2B histones, the H3 an d H4 polypeptides are properly folded only when assembled as H3-H4 dim ers or in higher-order histone assemblies. Therefore, coupling along t he interfaces of the two chains within the heterodimer is the major fa ctor contributing to the stabilization of the secondary and tertiary s tructures of the chains as well as of the histone dimers.