Semisynthesis of Ubiquitinated Histone H2B via Native Chemical Ligation

Abstract

The ubiquitylation of histone H2B at Lys (K) 120 is known to be associated with methylation of H3K4 and H3K79, transcription elongation, and DNA repair. Mechanistic studies of interactions between H2BK120ub and other chromatin-associated proteins, as well investigations of the role of ubiquitylation in chromatin function require milligram scale production of H2BK120ub. However, the direct synthesis of H2BK120ub by solid-phase peptide synthesis (SPPS) has not been possible due to limitations in yields of SPPS for long proteins. In this thesis, truncated H2B(1-116) and Ub(1-75) C-terminal α-thioesters were purified by intein fusion. An H2B(117-125) peptide fragment with an Ala117 to Cys mutation and acetamidomethyl protected Cys, Cys(Acm), attached to Lys120 was synthesized by SPPS. Finally, semisynthesis of H2BK120ub was performed by native chemical ligation (NCL) between various thioester fragments. The high purity of semisynthetic H2BK120ub was confirmed by ESI-MS and 15% SDS-PAGE, the ubiquitylated histone was used for octamer and mononucleosome reconstitution. The ubiquitylated mononucleosomes will be tested in numerous biological assays in the future to understand how H2BK120ub influences the function of human chromatin.