title: Exploring Nucleosome Unwrapping Using DNA Origami.
abstract: We establish a DNA origami based tool for quantifying conformational equilibria of biomolecular assemblies as a function of environmental conditions. As first application, we employed the tool to study the salt-induced disassembly of nucleosome core particles. To extract binding constants and energetic penalties, we integrated nucleosomes in the spectrometer such that unwrapping of the nucleosomal template DNA, leading from bent to more extended states was directly coupled to the conformation of the spectrometer. Nucleosome unwrapping was induced by increasing the ionic strength. The corresponding shifts in conformation equilibrium of the spectrometer were followed by direct conformation imaging using negative staining TEM and by FRET read out after gel electrophoretic separation of conformations. We find nucleosome dissociation constants in the picomolar range at low ionic strength (11 mM MgCl2), in the nanomolar range at intermediate ionic strength (11 mM MgCl2with 0.5-1 M NaCl) and in the micromolar range at larger ionic strength (11 mM MgCl2with ≥1.5 M NaCl). Integration of up to four nucleosomes stacked side-by-side, as it might occur within chromatin fibers, did not appear to affect the salt-induced unwrapping of nucleosomes. Presumably, such stacking interactions are already effectively screened at the nucleosome unwrapping conditions. Our spectrometer provides a modular platform with a direct read out to study conformational equilibria for targets from small biomolecules up to large macromolecular assemblies.
authors.forename: Jonas J
authors.initials: JJ
authors.lastname: Funke
authors.forename: Philip
authors.initials: P
authors.lastname: Ketterer
authors.affiliation: Biomedical Center, Molecular Biology, LMU Munich , 82152 Martinsried near Munich, Germany.
authors.forename: Corinna
authors.initials: C
authors.lastname: Lieleg
authors.affiliation: Biomedical Center, Molecular Biology, LMU Munich , 82152 Martinsried near Munich, Germany.
authors.forename: Philipp
authors.initials: P
authors.lastname: Korber
authors.affiliation: Center for Integrated Protein Science , 81377 Munich, Germany.
authors.forename: Hendrik
authors.initials: H
authors.lastname: Dietz
mesh_terms: D000818:Animals; D004247:DNA / Q000737:chemistry*; D004331:Drosophila melanogaster; D004625:Embryo, Nonmammalian; D031541:Fluorescence Resonance Energy Transfer; D006657:Histones; D046911:Macromolecular Substances; D009690:Nucleic Acid Conformation; D009707:Nucleosomes / Q000737:chemistry*; D009994:Osmolar Concentration
chemical_list: D006657:Histones; D046911:Macromolecular Substances; D009707:Nucleosomes; D004247:DNA
keywords: DNA origami; FRET; TEM; force spectroscopy; nucleosome