Ubiquitin (UB) is a 76-residue protein that is attached to other proteins in the cell to regulate a myriad of biological activities. There are seven lysine residues in UB, and each of them can be conjugated to another UB to form UB chains of diverse linkages. The complex cellular signals are encoded in the linkages of UB chains attached to cellular proteins. UB is transferred through an E1-E2-E3 cascade to cellular targets with E2, the UB conjugating enzyme, playing a key role in defining the linkage specificity of UB chains. Little is known about how E2s specifies the linkage types of the UB chains. To solve this problem, we replaced various Lys residues on UB with an unnatural amino acid N-L-thiapropyl-L-Lys (ThzK). We then used the ThzK residue at various Lys positions as a reactive handle to generate diUB probes of defined linkages by expressed peptide ligation. We are in progress to conjugate diUB conjugates of specific linkages to E2 and E3 enzymes to elucidate their mechanisms of action in UB chain assembly. We also show reactivity of the diUB probes with various DUBs that reverse the roles of deubiquitination.

Elucidating the mechanisms of ubiquitin (UB) chain assembly by di-UB probes enabled by unnatural amino acid incorporation

ACS Fall 2021

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.