Thermodynamic Stability of Archaeal Histones

Abstract

The temperature, salt, and pH dependencies of unfolding of four recombinant (r) archaeal histones (rHFoB from the mesophile Methanobacterium formicicum, and rHMfA, rHMfB, and rHPyA1 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 Tm 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. rHPyA1 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 1 M protein solution (T degree 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 Tmax values relative to the stability curve for rHFoB. The maximal free energies of unfolding for rHFoB, rHMfA, rHMfB, and rHPyA1 are 7.2, 15.5, 14.6, and 17.2 kcal/mol at 32, 35, 40, and 44 degrees C, respectively. T degree values for rHFoB, rHMfA, rHMfB, and rHPyA1 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.

Role of residual structure in the unfolded state of a thermophilic protein

“…Of the five thermodynamically characterized thermophile-mesophile protein pairs, four thermophilic proteins have up-shifted and broadened stability curves compared with their mesophilic homologs (8,(22)(23)(24)(25). It is difficult to make conclusions based on this limited data set, but it is possible that a large group of enzymes and other proteins requiring a finely tuned energy landscapes use lower ⌬Cp°values to balance a high melting temperature with the thermodynamic stability needed for optimal activity.…”

Archaeal Histone Tetramerization Determines DNA Affinity and the Direction of DNA Supercoiling

“…Synthesis of the recombinant archaeal histones, archaeal histone variants, and archaeal histone fusions was induced by addition of 400 M isopropyl-␤-D-thiogalactopyranoside to growing E. coli cultures. The proteins synthesized were purified, quantitated, and their CD spectra measured using an AVIV 62A-DA spectropolarimeter (AVIV, Lakewood, NJ) as previously described (32). The CD spectra obtained for HMfA, HMfB, the HMfB variants, and the histone fusions were almost identical (available on request), consistent with very similar folded native structures.…”

A thermodynamic study of mesophilic, thermophilic, and hyperthermophilic l‐arabinose isomerases: The effects of divalent metal ions on protein stability at elevated temperatures

“…However, no universal basis of stability has been recognized because the stability of different enzymes has different origins. For this reason, more extensive studies have been performed to elucidate the fundamentals of protein stability in terms of macromolecular interactions based on thermodynamics [10][11][12].…”