Proteomics

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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

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Dataset's files

Source:
Action DRS
10mg_eAf_1_EThcD.csv Csv
10mg_eAf_1_HCD.csv Csv
10mg_eAf_2_EThcD.csv Csv
10mg_eAf_2_HCD.csv Csv
10mg_eAf_3_EThcD.csv Csv
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Publications

Protein ADP-ribosylation is a reversible post-translational modification that regulates important cellular functions. The identification of modified proteins has proven challenging and has mainly been achieved via enrichment methodologies. Random mutagenesis was used here to develop an engineered Af1521 ADP-ribose binding macro domain protein with 1000-fold increased affinity towards ADP-ribose. The crystal structure reveals that two point mutations K35E and Y145R form a salt bridge within the A  ...[more]

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