Site-specific <i>in vivo</i> protein SUMOylation <i>via</i> translational incorporation of a proximity-reactive pyrrolysine analogue.

ABSTRACT: Here, we present a novel strategy that integrates genetic-code expansion and proximity-induced crosslinking to achieve site-specific in vivo SUMOylation. This approach involves incorporating the unnatural amino acid 2-chloroacetyl-Nε-lysine (ClAcK) into the target protein using MmFAcKRS1, a previously reported pyrrolysyl-tRNA synthetase mutant that we have repurposed for ClAcK incorporation. Once incorporated, ClAcK can be specifically targeted to react with a cysteine engineered at the C-terminus of SUMO variants leading to a chemically SUMOylated protein. This reaction is proximity-induced, and preferentially promoted when the two reactive groups are in close spatial proximity. We therefore leverage the natural affinity of SUMO for SUMO-interacting motifs (SIMs) on target p

SUBMITTER: Chan YH 

PROVIDER: S-EPMC11791516 | biostudies-literature | 2025 Mar

REPOSITORIES: biostudies-literature