title: 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.
abstract: 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 (approximately 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 approximately 50% of the secondary structure and approximately 60% of the surface area burial of the native dimer. The stability of the dimeric intermediate is approximately 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, alpha-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.
authors.affiliation: School of Molecular Biosciences, Washington State University, Pullman, WA 99164-4660, USA.
authors.forename: Brandon J
authors.initials: BJ
authors.lastname: Placek
authors.forename: Lisa M
authors.initials: LM
authors.lastname: Gloss
mesh_terms: D019281:Dimerization; D006657:Histones / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D011489:Protein Denaturation; D017510:Protein Folding; D020836:Protein Structure, Quaternary / Q000187:drug effects; D014508:Urea / Q000494:pharmacology
chemical_list: D006657:Histones; D014508:Urea