Method for the synthesis of mono-ADP-ribose conjugated peptides.
ADP-ribosylation is an important post-translational modification involved in processes including cellular replication, DNA repair, and cell death. Despite these roles, the functions of ADP-ribosylation, in particular mono-ADP-ribosylation, remain poorly understood. The development of a technique to generate large amounts of site-specific, ADP-ribosylated peptides would provide a useful tool for deconvoluting the biochemical roles of ADP-ribosylation. Here we demonstrate that synthetic histone H2B tail peptides, incorporating aminooxy or N-methyl aminooxy functionalized amino acids, can be site-specifically conjugated to ADP-ribose. These peptides are recognized as substrates by the ADP-ribosylation biochemical machinery (PARP1), can interact with the ADP-ribose binding proteins macroH2A1.1 and PARP9, and demonstrate superior enzymatic and chemical stability when compared to ester-linked ADP-ribose. In addition, the incorporation of benzophenone photo-cross-linkers into these peptides is demonstrated to provide a means to probe for and enrich ADP-ribose binding proteins.
Laboratory of Synthetic Protein Chemistry, The Rockefeller University, New York, New York 10065, USA.
Peter M
PM
Moyle
Tom W
TW
Muir
D000246:Adenosine Diphosphate Ribose / Q000737:chemistry* / Q000378:metabolism; D001710:Biotin / Q000737:chemistry; D006367:HeLa Cells; D006657:Histones / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D019151:Peptide Library; D010455:Peptides / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D013379:Substrate Specificity
D006657:Histones; D019151:Peptide Library; D010455:Peptides; D000246:Adenosine Diphosphate Ribose; D001710:Biotin