THERMODYNAMIC STABILITY OF ARCHAEAL HISTONES

The temperature, salt, and pH dependencies of unfolding of four recomb inant (r) archaeal histones (rHFoB from the mesophile Methanobacterium formicicum, and rHMfA, rHMfB, and rHPyAl from the hyperthermophiles M ethanothermus fervidus and Pyrococcus strain GB-3a) have been determin ed by circular dichroism spectroscopy (CD) and differential scanning c alorimetry (DSC). The thermal unfolding of these proteins is >90% reve rsible, with concentration-dependent apparent T-m values and asymmetri c unfolding transitions that are fit well by a two-state unfolding mod el in which a histone dimer unfolds to two random coil monomers. rHPyA l 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 concentrati ons 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 p arameters correlate with the growth temperature of the originating arc haeon. The changes in heat capacity upon unfolding are similar for the four histones, indicating that enhanced thermostability is not obtain ed by altering the curvature of the stability curve. Rather, the stabi lity curves for the histones from the hyperthermophiles are displaced vertically to higher energies and laterally to higher T-max values rel ative to the stability curve for rHFoB. The maximal free energies of u nfolding 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 wit hin the conserved histone fold that might confer these stability diffe rences are discussed.