Folding mechanism of the (H3-H4)2 histone tetramer of the core nucleosome.
To further understand oligomeric protein assembly, the folding and unfolding kinetics of the H3-H4 histone tetramer have been examined. The tetramer is the central protein component of the core nucleosome, which is the basic unit of DNA compaction into chromatin in the eukaryotic nucleus. This report provides the first kinetic folding studies of a protein containing the histone fold dimerization motif, a motif observed in several protein-DNA complexes. Previous equilibrium unfolding studies have demonstrated that, under physiological conditions, there is a dynamic equilibrium between the H3-H4 dimer and tetramer species. This equilibrium is shifted predominantly toward the tetramer in the presence of the organic osmolyte trimethylamine-N-oxide (TMAO). Stopped-flow methods, monitoring intrinsic tyrosine fluorescence and far-UV circular dichroism, have been used to measure folding and unfolding kinetics as a function of guanidinium hydrochloride (GdnHCl) and monomer concentrations, in 0 and 1 M TMAO. The assignment of the kinetic phases was aided by the study of an obligate H3-H4 dimer, using the H3 mutant, C110E, which destabilizes the H3-H3' hydrophobic four-helix bundle tetramer interface. The proposed kinetic folding mechanism of the H3-H4 system is a sequential process. Unfolded H3 and H4 monomers associate in a burst phase reaction to form a dimeric intermediate that undergoes a further, first-order folding process to form the native dimer in the rate-limiting step of the folding pathway. H3-H4 dimers then rapidly associate with a rate constant of > or =10(7) M(-1)sec(-1) to establish a dynamic equilibrium between the fully assembled tetramer and folded H3-H4 dimers.
Banks
Douglas D
DD
School of Molecular Biosciences, Washington State University, Pullman, Washington 99164-4660, USA.
Gloss
Lisa M
LM
D002942:Circular Dichroism; D004268:DNA-Binding Proteins / Q000032:analysis / Q000737:chemistry; D019281:Dimerization; D019791:Guanidine / Q000737:chemistry; D006657:Histones / Q000032:analysis / Q000737:chemistry*; D007700:Kinetics; D008856:Microscopy, Fluorescence; D009707:Nucleosomes / Q000737:chemistry*; D011487:Protein Conformation; D017510:Protein Folding
D004268:DNA-Binding Proteins; D006657:Histones; D009707:Nucleosomes; D019791:Guanidine