Engineering Af1521 macro domain increases ADP-ribose binding affinity and leads to the identification of more ADP-ribosylated proteins

ABSTRACT: Protein ADP-ribosylation is a covalent, reversible post-translational modification that has important functions in several cellular mechanisms. The identification of modified proteins in cells has proven challenging and, due to the low abundance of protein ADP-ribosylation, has mainly been possible via enrichment methodologies using ADP-ribose binding domains. Here, using random mutagenesis and in vitro selection with an ADP-ribosylated histone peptide, we engineered the archaeal Af1521 macro domain to generate an engineered isoform containing nine mutations that displays significantly increased affinity towards ADP-ribose. Comparison of the wild-type and the engineered Af1521 macro domains using our proteomic ADP-ribosylome enrichment workflow demonstrated an increased identification rate of ADP-ribosylated proteins with the engineered Af1521.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell

SUBMITTER: Kathrin Nowak  

LAB HEAD: Michael O. Hottiger

PROVIDER: PXD016686 | Pride | 2020-10-20

REPOSITORIES: Pride