Thermodynamic stability of archaeal histones

The temperature, salt, and pH dependencies of unfolding of four recombinant (r) archaeal histones (rHFoB from the mesophile Methanobacterium formicicum, and rHMfA, rHMfB, and rHPyAl from the hyperthermophiles Methanothermus fervidus and Pyrococcus strain GB-3a) have been determined by circular dichroism spectroscopy (CD) and differential scanning calorimetry (DSC). The thermal unfolding of these proteins is >90% reversible, with concentration-dependent apparent T-m values and asymmetric unfolding transitions that are fit well by a two-state unfolding model in which a histone dimer unfolds to two random coil monomers. rHPyAl dimers are stable in the absence of salt, whereas rHMfA, rHMfB, and rHFoB dimers unfold at 20 degrees C and pH 2 in solutions containing <200 mM, <400 mM, and <1.5 M KCl, respectively, rHMfA, rHMfB, and rHFoB also experience significant cold denaturation in low salt concentrations and at low pH. The midpoint of thermal unfolding of a I M protein solution (T degrees value) and the temperature dependency of the free energy of unfolding have been established for each histone, and both parameters correlate with the growth temperature of the originating archaeon. The changes in heat capacity upon unfolding are similar for the four histones, indicating that enhanced thermostability is not obtained by altering the curvature of the stability curve. Rather, the stability curves for the histones from the hyperthermophiles are displaced vertically to higher energies and laterally to higher T-max values relative to the stability curve for rHFoB. The maximal free energies of unfolding for rHFoB, rHMfA, rHMfB, and rHPyAl are 7.2, 15.5, 14.6, and 17.2 kcal/mol at 32, 35, 40, and 44 degrees C, respectively. T degrees values for rHFoB, rHMfA, rHMfB, and rHPyAl are 75, 104, 113, and 114 degrees C, respectively, at pH 5 in 0.2 M KCl. Structural features within the conserved histone fold that might confer these stability differences are discussed.