title: Preparation, Biochemical Analysis, and Structure Determination of Methyllysine Readers.
abstract: In-depth in vitro characterization of methyllysine reader domains and their association with cognate methyllysine substrates is essential to better understand fundamental mechanisms of chromatin regulation and to design targeted therapeutics that disrupt these interactions. In this chapter, we summarize commonly used methods for preparation, biochemical characterization, and determination of structures of methyllysine reader domains. We provide a detailed protocol for the preparation of a GST-tagged methyllysine reader domain and for analysis of histone-binding activities using a combination of pull-down, tryptophan fluorescence, and NMR assays, and describe initial steps toward crystallization of the complexes.
authors.affiliation: University of Iowa, Iowa City, IA, United States. Electronic address: catherine-musselman@uiowa.edu.
authors.forename: C A
authors.initials: CA
authors.lastname: Musselman
authors.affiliation: University of Colorado School of Medicine, Aurora, CO, United States. Electronic address: tatiana.kutateladze@ucdenver.edu.
authors.forename: T G
authors.initials: TG
authors.lastname: Kutateladze
mesh_terms: D000818:Animals; D018360:Crystallography, X-Ray / Q000379:methods; D044127:Epigenesis, Genetic; D004926:Escherichia coli / Q000235:genetics; D005982:Glutathione Transferase / Q000235:genetics; D042421:Histone Code; D006657:Histones / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D008239:Lysine / Q000031:analogs & derivatives* / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D008745:Methylation; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D010957:Plasmids / Q000235:genetics; D000072417:Protein Domains; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015854:Up-Regulation
chemical_list: D006657:Histones; D011993:Recombinant Fusion Proteins; C049907:2-methyllysine; D005982:Glutathione Transferase; D008239:Lysine
keywords: Epigenetics; Histone; Methyllysine; Protein purification; Reader; Structure