4339883

Activated cysteine-based protein ligation to Functiionalize ubiquitin and ubiquitin-like proteins as activity-based protein profiling probes

Date
August 19, 2025

To functionalize a protein at its C-terminus for ligation with other chemical components,
we showed that a cysteine in a recombinant protein was chemically activated by a small molecule
cyanylating reagent for exchange with small molecule amines including a number of L- and D-
amino acids and hydrazine. We demonstrated the versatility of this activated cysteine-directed protein ligation (ACPL) approach with the successful synthesis of ubiquitin conjugates and ubiquitin-like protein conjugates. Using this new approach, we demonstrated that ubiquitin aza peptide esters were readily generated and served as novel activity-based protein probes to profile enzymes involved in the ubiquitin-proteasome pathway. Similarly, we used ACPL to synthesize an active-based probe based on URM1 that is a ubiquitin-like protein. Applying this probe for proteomic characterizations of enzymes involved in the URM1 pathway identified multiple regulatory enzymes that have never been reported.

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DIVISION/COMMITTEE: [BIOL: Division of Biochemistry and Chemical Biology]