title: Dual tagging as an approach to isolate endogenous chromatin remodeling complexes from Saccharomyces cerevisiae.
abstract: Affinity isolation has been an essential technique for molecular studies of cellular assemblies, such as the switch/sucrose non-fermentable (SWI/SNF) family of ATP-dependent chromatin remodeling complexes. However, even biochemically pure isolates can contain heterogeneous mixtures of complexes and their components. In particular, purification strategies that rely on affinity tags fused to only one component of a complex may be susceptible to this phenomenon. This study demonstrates that fusing purification tags to two different proteins enables the isolation of intact complexes of remodels the structure of chromatin (RSC). A Protein A tag was fused to one of the RSC proteins and a Twin-Strep tag to another protein of the complex. By mass spectrometry, we demonstrate the enrichment of the RSC complexes. The complexes had an apparent Svedberg value of about 20S, as shown by glycerol gradient ultracentrifugation. Additionally, purified complexes were demonstrated to be functional. Electron microscopy and single-particle analyses revealed a conformational rearrangement of RSC upon interaction with acetylated histone H3 peptides. This purification method is useful to purify functionally active, structurally well-defined macromolecular assemblies.
authors.affiliation: Department of Biomedicine, Aarhus University, Wilhelm Meyers Allé 3, Building 1233/1234, DK-8000 Aarhus C, Denmark.
authors.forename: Tzong-Yuan
authors.initials: TY
authors.lastname: Lin
authors.affiliation: Department of Biomedicine, Aarhus University, Wilhelm Meyers Allé 3, Building 1233/1234, DK-8000 Aarhus C, Denmark.
authors.forename: Andriy
authors.initials: A
authors.lastname: Voronovsky
authors.affiliation: Bioanalytical Mass Spectrometry Group, Max-Planck-Institute for Biophysical Chemistry, Am Fassberg 11, D-37077 Göttingen, Germany.
authors.forename: Monika
authors.initials: M
authors.lastname: Raabe
authors.affiliation: Bioanalytical Mass Spectrometry Group, Max-Planck-Institute for Biophysical Chemistry, Am Fassberg 11, D-37077 Göttingen, Germany; Bioanalytics, Institute for Clinical Chemistry, University Medical Center Göttingen, Robert-Koch-Strasse 40, D-37075 Göttingen, Germany.
authors.forename: Henning
authors.initials: H
authors.lastname: Urlaub
authors.affiliation: Stereology and EM Laboratory, Department of Clinical Medicine, Aarhus University, Wilhelm Meyers Allé 3, Building 1233/1234, DK-8000 Aarhus C, Denmark; Centre for Stochastic Geometry and Advanced Bioimaging, Aarhus University, Wilhelm Meyers Allé 3, Building 1233/1234, DK-8000 Aarhus C, Denmark.
authors.forename: Bjoern
authors.initials: B
authors.lastname: Sander
authors.affiliation: Department of Biomedicine, Aarhus University, Wilhelm Meyers Allé 3, Building 1233/1234, DK-8000 Aarhus C, Denmark; Centre for Stochastic Geometry and Advanced Bioimaging, Aarhus University, Wilhelm Meyers Allé 3, Building 1233/1234, DK-8000 Aarhus C, Denmark. Electronic address: mgolas@biomed.au.dk.
authors.forename: Monika M
authors.initials: MM
authors.lastname: Golas
mesh_terms: D002843:Chromatin / Q000235:genetics* / Q000302:isolation & purification*; D042002:Chromatin Assembly and Disassembly / Q000235:genetics*; D002846:Chromatography, Affinity / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D013194:Staining and Labeling / Q000379:methods
chemical_list: D002843:Chromatin
keywords: Chromatin remodeling; Dual tagging; Purification; RSC complex; SWI/SNF family; Saccharomyces cerevisiae