title: Protein-protein Förster resonance energy transfer analysis of nucleosome core particles containing H2A and H2A.Z.
abstract: A protein-protein Förster resonance energy transfer (FRET) system, employing probes at multiple positions, was designed to specifically monitor the dissociation of the H2A-H2B dimer from the nucleosome core particle (NCP). Tryptophan donors and Cys-AEDANS acceptors were chosen because, compared to previous NCP FRET fluorophores, they: (1) are smaller and less hydrophobic, which should minimize perturbations of histone and NCP structure; and (2) have an R0 of 20 A, which is much less than the dimensions of the NCP (approximately 50 A width and approximately 100 A diameter). Equilibrium protein unfolding titrations indicate that the donor and acceptor moieties have minimal effects on the stability of the H2A-H2B dimer and (H3-H4)2 tetramer. NCPs containing the various FRET pairs were reconstituted with the 601 DNA positioning element. Equilibrium NaCl-induced dissociation of the modified NCPs showed that the 601 sequence stabilized the NCP to dimer dissociation relative to weaker positioning sequences. This finding implies a significant role for the H2A-H2B dimers in determining the DNA sequence dependence of NCP stability. The free energy of dissociation determined from reversible and well-defined sigmoidal transitions revealed two distinct phases reflecting the dissociation of individual H2A-H2B dimers, confirming cooperativity as suggested previously; these data allow quantitative description of the cooperativity. The FRET system was then used to study the effects of the histone variant H2A.Z on NCP stability; previous studies have reported both destabilizing and stabilizing effects. H2A.Z FRET NCP dissociation transitions suggest a slight increase in stability but a significant increase in cooperativity of the dimer dissociations. Thus, the utility of this protein-protein FRET system to monitor the effects of histone variants on NCP dynamics has been demonstrated, and the system appears equally well-suited for dissection of the kinetic processes of dimer association and dissociation from the NCP.
authors.affiliation: School of Molecular Biosciences, Washington State University, Pullman, WA 99164-4660, USA.
authors.forename: Duane A
authors.initials: DA
authors.lastname: Hoch
authors.forename: Jessica J
authors.initials: JJ
authors.lastname: Stratton
authors.forename: Lisa M
authors.initials: LM
authors.lastname: Gloss
mesh_terms: D000818:Animals; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry; D019281:Dimerization; D004735:Energy Transfer; D031541:Fluorescence Resonance Energy Transfer; D006657:Histones / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D009707:Nucleosomes / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D013816:Thermodynamics; D014364:Tryptophan / Q000737:chemistry; D014982:Xenopus laevis
chemical_list: D006657:Histones; D009707:Nucleosomes; D014364:Tryptophan; D003545:Cysteine