Equilibrium stabilities of the histone oligomers and kinetic folding mechanism of the H3 -H4 tetramer

The stabilities of the structurally related histone oligomers were compared to aid in understanding the thermodynamics of nucleosome assembly. The equilibrium stabilities of recombinant wild type H3-H4 tetramer and H2A-H2B dimer have been determined by guanidinium-induced denaturation, using fluorescence and circular dichroism (CD) spectroscopies. The unfolding of the H2A-H2B dimer is a two-state process, with no detected equilibrium intermediates. The H3-H4 tetramer is unstable at moderate ionic strengths (mu ∼ 0.2 M). TMAO (trimethylamine-N-oxide) was used to stabilize the tetramer; the stability of the H2A-H2B dimer was determined under the same solvent conditions. The equilibrium unfolding of H3-H4 is best described by a three-state mechanism, with well-folded H3-H4 dimers as a populated intermediate. Compared to H2A-H2B, the H3-H3 tetramer interface and the H3-H4 histone fold are strikingly less stable. The free energy of unfolding, in the absence of denaturant, for the H3-H4 and H2A-H2B dimers are 12.4 and 21.0 kcal mol-1, respectively, in 1 M TMAO. Stopped-flow fluorescence and CD were used to determine the folding mechanism of the H3-H4 tetramer. The guanidinium and protein concentration dependencies of the unfolding and folding kinetic reactions were determined in the presence and absence of 1 M TMAO. The kinetic folding mechanism is a sequential process: (1) unfolded H3 and H4 monomers associate in a burst phase reaction to form a dimeric intermediate; (2) this intermediate further folds in a first-order reaction to yield the native dimer in the rate-limiting step of the folding reaction. H3-H4 dimers then rapidly associate to form the tetramer, establishing a dynamic equilibrium between folded dimers and the fully assembled tetramer.