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Three-state Kinetic Folding Mechanism of the H2A/H2B Histone Heterodimer: the N-terminal Tails Affect the Transition State Between a Dimeric Intermediate and the Native Dimer

Journal of Molecular Biology
Publication Date
DOI: 10.1016/j.jmb.2004.11.006
  • Kinetics
  • Circular Dichroism
  • Fluorescence
  • Stopped-Flow
  • Biology


The H2A/H2B heterodimer is a component of the nucleosome core particle, the fundamental repeating unit of chromatin in all eukaryotic cells. The kinetic folding mechanism for the H2A/H2B dimer has been determined from unfolding and refolding kinetics as a function of urea using stopped-flow, circular dichroism and fluorescence methods. The kinetic data are consistent with a three-state mechanism: two unfolded monomers associate to form a dimeric intermediate in the dead-time of the SF instrument (∼5 ms); this intermediate is then converted to the native dimer by a slower, first-order reaction. Analysis of the burst-phase amplitudes as a function of denaturant indicates that the dimeric kinetic intermediate possesses ∼50% of the secondary structure and ∼60% of the surface area burial of the native dimer. The stability of the dimeric intermediate is ∼30% of that of the native dimer at the monomer concentrations employed in the SF experiments. Folding-to-unfolding double-jump experiments were performed to monitor the formation of the native dimer as a function of folding delay times. The double-jump data demonstrate that the dimeric intermediate is on-pathway and obligatory. Formation of a transient dimeric burst-phase intermediate has been observed in the kinetic mechanism of other intertwined, segment-swapped, α-helical, DNA-binding dimers, such as the H3–H4 histone dimer, Escherichia coli factor for inversion stimulation and E. coli Trp repressor. The common feature of a dimeric intermediate in these folding mechanisms suggests that this intermediate may accelerate protein folding, when compared to the folding of archael histones, which do not populate a transient dimeric species and fold more slowly.

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