Rapid Semisynthesis of Acetylated and Sumoylated Histone Analogs

Abstract

The density and diversity of post-translational modifications (PTMs) observed in histone proteins typically limits 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 Lys 14 or Lys 56, using thiol-ene coupling chemistry. Both strategies afford multi-milligram 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.

Nucleosome conformation dictates the histone code

“…For acetyl lysine analog, 15 N-labeled histone H4 with a K-to-C mutation at lysine 16 was expressed and purified similar to wild-type H4 as described above. The acetyl-mimetic residue (K s 16ac) was generated through radical-mediated thiol-ene addition to the thiol group of cysteine as previously described ( Li et al, 2011 ; Bryan et al, 2017 ; Dhall et al, 2016 ; Dhall et al, 2014 ). Briefly, lyophilized H4K16C protein was resuspended to a concentration of 1 mM in de-gassed reaction buffer (a 200 mM acetic acid and 15 mM L-glutathione solution, pH 4.0 to which 5 mM azo radical initiator VA-044 ( Wako chemicals #27776-21-2), and 50 mM N-vinylacetamide ( TCI #5202-78-8) are added immediately before the reaction).…”

Strategies for Generating Modified Nucleosomes: Applications within Structural Biology Studies

“…Thus, several approaches have been explored for chemical modification of cysteine in order to generate modification analogues at specific positions in histones. Direct alkylation of cysteine has been successfully used to install mono-, di-, and tri-methyl lysine, acetyl-lysine, GlcNAc-serine, and methyl-arginine analogues [34][35][36][37][38] . More recently, a new cysteine modification strategy was employed leading to installation of hydrazide analogues to generate acetyl-lysine, 2hydroxyisobutyryl-lysine, and ubiquityl-lysine analogues 39 .…”

“…Thus, several approaches have been explored for chemical modification of cysteine to generate modification analogues at specific positions in histones. Direct alkylation of cysteine has been successfully used to install mono-, di-, and trimethyl lysine, acetyl-lysine, GlcNAc-serine, and methyl-arginine analogues. − More recently, a new cysteine modification strategy was employed leading to installation of hydrazide analogues to generate acetyl-lysine, 2-hydroxyisobutyryl-lysine, and ubiquityl-lysine analogues . The analogue approach is advantageous over NCL/EPL and genetic installation as it is possible to generate large quantities of the modified histone relatively inexpensively and is generally straightforward to carry out.…”

Ubiquitin Signaling: Chemistry Comes to the Rescue

“…Since the connectivity between the Ub-like modifiers and the protein substrate is also via an isopeptide bond, the lessons learned from the synthesis of various Ub conjugates could be applied to the preparation of proteins decorated with Ub-like modifiers to decipher their role in different cellular events and associated diseases. Indeed, several laboratories are already pursuing these directions, and exciting results from these studies will surely emerge. − …”