A facile method to synthesize histones with posttranslational modification mimics

Abstract

Using an evolved pyrrolysyl-tRNA synthetase-tRNA(Pyl) pair, a Se-alkylselenocysteine was genetically incorporated into histone H3 with a high protein expression yield. Quantitative oxidative elimination of Se-alkylselenocysteine followed by Michael addition reactions with various thiol nucleophiles generated biologically active mimics of H3 with posttranslational modifications including lysine methylation, lysine acetylation, and serine phosphorylation.

Figure 1

The site-specific incorporation of 1, 2, and 3 at the K9 position of H3. Proteins were expressed in BL21 cells transformed with pEVOL-mKRS1-pylT and pET-H3K9TAG in LB medium supplemented with 2 mM 1, 2 mM 2, or 1.5 mM 3.

Figure 2

(A) Immunoprecipitation assay of wild type H3, H3K9m¹sK, and H3K9m²sK using HP-1β. Each protein was incubated with HP-1β and anti-HP-1β antibody and pulled down by immobilized protein A resins that bind Fc region of anti-HP-1β. PD represents pull down. (B) Immunoprecipitation assay of wild type H3 and H3K9m³sK using HP-1β. (C) Western blot analysis of wild type H3 and H3K9AcsK probed by anti-H3K9Ac antibody.

Scheme 1

Oxidative elimination reactions to generate Dha that undergoes Michael addition reactions to form different posttranslational modification mimics.