Site-specific ubiquitylation and SUMOylation using genetic-code expansion and sortase

Phosphine‐Activated Lysine Analogues for Fast Chemical Control of Protein Subcellular Localization and Protein SUMOylation

“…SUMOylation affects protein function in a variety of ways, including localization and stability . Although several approaches for site‐specifically SUMOylating proteins have arisen, and several investigators have globally controlled SUMOylation or turned it off at specific sites by mutating the acceptor lysine to arginine, conditional turn‐on of native SUMOylation sites in live cells with wild‐type (WT) SUMO1 has not been reported to the best of our knowledge.…”

“…While small molecules, siRNAs, proteins, and genetic approaches have been validated to turn off RanGAP1 SUMOylation with varying degrees of specificity, approaches for selective SUMOylation turn‐on remain rare and require either SUMO overexpression, mutations to the SUMO modifier, or may only be carried out in test tubes as opposed to live cells . We addressed this methodology gap through development of small‐molecule‐triggered SUMOylation via the endogenous conjugation machinery, an approach that is applicable to other post‐translational modifications (PTMs) as well.…”

“…Among bioorthogonal reactions, the Staudinger reduction of aryl azides by phosphines is highly compatible with live cells and organisms . Both functional groups are abiotic.…”

Photo‐induced and Rapid Labeling of Tetrazine‐Bearing Proteins via Cyclopropenone‐Caged Bicyclononynes

“…Furthermore,s fGFP bearing BocK at position 150 instead of mTetK did not result in any labeling,confirming the selectivity of photo-iEDDAC (Supporting Information, Figure S9). To check how DMBO compounds performed in SPAACr eactions,w ei ncubated an azidebearing sfGFP [25] with photo-11.A lso this labeling took 2-3hours to go to completion, highlighting the superior reactivity of DMBO in iEDDAC versus SPAAC(Supporting Information, Figure S11). To check how DMBO compounds performed in SPAACr eactions,w ei ncubated an azidebearing sfGFP [25] with photo-11.A lso this labeling took 2-3hours to go to completion, highlighting the superior reactivity of DMBO in iEDDAC versus SPAAC(Supporting Information, Figure S11).…”

“…Also,T etK-modified proteins (sfGFP-1-His6) reacted selectively upon light-activation with photo-11,a lbeit much more slowly,a nd quantitative protein labeling with 25-fold excess of 11 was only achieved after several hours of incubation (Supporting Information, Figure S10). To check how DMBO compounds performed in SPAACr eactions,w ei ncubated an azidebearing sfGFP [25] with photo-11.A lso this labeling took 2-3hours to go to completion, highlighting the superior reactivity of DMBO in iEDDAC versus SPAAC(Supporting Information, Figure S11). Furthermore,a zide-modified amino acids are not completely stable towards reduction to the corresponding amine in the cytosol of E. coli and therefore our stable methyl-tetrazine amino acids make for more reliable reporters in live E. coli.…”