title: Single-pair fluorescence resonance energy transfer of nucleosomes in free diffusion: optimizing stability and resolution of subpopulations.
abstract: We applied fluorescence detection methods on the single-molecule level to study structural variations and dynamic processes occurring within nucleosomes. Four fluorescent nucleosome constructs were made by attaching donor and acceptor fluorophores to different positions of two nucleosome positioning sequences and reconstituting nucleosomes by salt dialysis. The photochemical and biochemical stability of nucleosomes under single-molecule conditions was optimized by adding inert protein and free radical capturing additives, allowing us to define the best experimental conditions for single-molecule spectroscopy on highly diluted solutions of nucleosome complexes. We could demonstrate for the first time the resolution of conformational subpopulations of nucleosomes by single-pair fluorescence resonance energy transfer in a freely diffusing system and could show the effect of thermally induced nucleosome repositioning.
authors.affiliation: Division of Biophysics of Macromolecules (B040), German Cancer Research Center, D-69120 Heidelberg, Germany.
authors.forename: Alexander
authors.initials: A
authors.lastname: Gansen
authors.forename: Florian
authors.initials: F
authors.lastname: Hauger
authors.forename: Katalin
authors.initials: K
authors.lastname: Tóth
authors.forename: Jörg
authors.initials: J
authors.lastname: Langowski
mesh_terms: D015342:DNA Probes / Q000737:chemistry; D004058:Diffusion; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D009707:Nucleosomes / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D012996:Solutions
chemical_list: D015342:DNA Probes; D009707:Nucleosomes; D012996:Solutions