title: Rapid Semisynthesis of Acetylated and Sumoylated Histone Analogs.
abstract: The density and diversity of posttranslational modifications (PTMs) observed in histone proteins typically limit their purification to homogeneity from biological sources. Access to quantities of uniformly modified histones is, however, critical for investigating the downstream effects of histone PTMs on chromatin-templated processes. Therefore, a number of semisynthetic methodologies have been developed to generate histones bearing precisely defined PTMs or close analogs thereof. In this chapter, we present two optimized and rapid strategies for generating functional analogs of site-specifically acetylated and sumoylated histones. First, we describe a convergent strategy to site-specifically attach the small ubiquitin-like modifier-3 (SUMO-3) protein to the site of Lys12 in histone H4 by means of a disulfide linkage. We then describe the generation of thialysine analogs of histone H3 acetylated at Lys14 or Lys56, using thiol-ene coupling chemistry. Both strategies afford multimilligram quantities of uniformly modified histones that are easily incorporated into mononucleosomes and nucleosome arrays for biophysical and biochemical investigations. These methods are readily extendable to any desired sites in the four core nucleosomal histones and their variant forms.
authors.affiliation: University of Washington, Seattle, WA, United States.
authors.forename: A
authors.initials: A
authors.lastname: Dhall
authors.affiliation: University of Washington, Seattle, WA, United States.
authors.forename: C E
authors.initials: CE
authors.lastname: Weller
authors.affiliation: University of Washington, Seattle, WA, United States. Electronic address: chatterjee@chem.washington.edu.
authors.forename: C
authors.initials: C
authors.lastname: Chatterjee
mesh_terms: D000107:Acetylation; D000818:Animals; D003001:Cloning, Molecular / Q000379:methods; D003545:Cysteine / Q000031:analogs & derivatives / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D006657:Histones / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D008239:Lysine / Q000031:analogs & derivatives / Q000737:chemistry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed / Q000379:methods; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D025841:Small Ubiquitin-Related Modifier Proteins / Q000737:chemistry* / Q000235:genetics; D058207:Sumoylation; D014452:Ubiquitins / Q000138:chemical synthesis / Q000737:chemistry / Q000235:genetics
chemical_list: D004220:Disulfides; D006657:Histones; D011994:Recombinant Proteins; C492189:SUMO3 protein, human; D025841:Small Ubiquitin-Related Modifier Proteins; D014452:Ubiquitins; C000204:S-2-aminoethyl cysteine; D008239:Lysine; D003545:Cysteine
keywords: Acetylation; Chromatin; Disulfide; Histone; SUMO; Sumoylation semisynthesis