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  Dual tagging as an approach to isolate endogenous chromatin remodeling complexes from Saccharomyces cerevisiae.

Lin, T. Y., Voronovsky, A., Raabe, M., Urlaub, H., Sander, B., & Golas, M. M. (2015). Dual tagging as an approach to isolate endogenous chromatin remodeling complexes from Saccharomyces cerevisiae. Biochimica et Biophysica Acta - Proteins and Proteomics, 1854(3), 198-208. doi:10.1016/j.bbapap.2014.11.009.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-0025-7666-6 Version Permalink: http://hdl.handle.net/11858/00-001M-0000-002A-1C04-3
Genre: Journal Article

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 Creators:
Lin, T. Y., Author
Voronovsky, A., Author
Raabe, M.1, Author              
Urlaub, H.1, Author              
Sander, B., Author
Golas, M. M., Author
Affiliations:
1Research Group of Bioanalytical Mass Spectrometry, MPI for biophysical chemistry, Max Planck Society, ou_578613              

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Free keywords: Chromatin remodeling; Dual tagging; Purification; RSC complex; SWI/SNF family; 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.

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Language(s): eng - English
 Dates: 2014-12-052015-03
 Publication Status: Published in print
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 Rev. Method: Peer
 Identifiers: DOI: 10.1016/j.bbapap.2014.11.009
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Title: Biochimica et Biophysica Acta - Proteins and Proteomics
Source Genre: Journal
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Pages: - Volume / Issue: 1854 (3) Sequence Number: - Start / End Page: 198 - 208 Identifier: -