Post-translational modification with the small ubiquitinlike modifier (SUMO) affects thousands of proteins in the human proteome and is implicated in numerous cellular processes. The main outcome of SUMO conjugation is a rewiring of protein−protein interactions through recognition of the modifier's surface by SUMO binding proteins. The SUMO-interacting motif (SIM) mediates binding to a groove on SUMO; however, the low affinity of this interaction and the poor conservation of SIM sequences complicates the isolation and identification of SIM proteins. To address these challenges, we have designed and biochemically characterized monomeric and multimeric SUMO-2 probes with a genetically encoded photo-cross-linker positioned next to the SIM binding groove. Following photoinduced covalent capture, even weak SUMO binders are not washed away during the enrichment procedure, and very stringent washing conditions can be applied to remove nonspecifically binding proteins. A total of 329 proteins were isolated from nuclear HeLa cell extracts and identified using mass spectrometry. We found the molecular design of our probes was corroborated by the presence of many established SUMO interacting proteins and the high percentage (>90%) of hits containing a potential SIM sequence, as predicted by bioinformatic analyses. Notably, 266 of the 329 proteins have not been previously reported as SUMO binders using traditional noncovalent enrichment procedures. We confirmed SUMO binding with purified proteins and mapped the position of the covalent cross-links for selected cases. We postulate a new SIM in MRE11, involved in DNA repair. The identified SUMO binding candidates will help to reveal the complex SUMO-mediated protein network.

The thiol group of the cysteine side chain is arguably the most versatile chemical handle in proteins. To expand the scope of established and commercially available thiol bioconjugation reagents, we genetically encoded a second such functional moiety in form of a latent thiol group that can be unmasked under mild physiological conditions. Phenylacetamidomethyl (Phacm) protected homocysteine (HcP) was incorporated and its latent thiol group unmasked on purified proteins using penicillin G acylase (PGA). The enzymatic deprotection depends on steric accessibility, but can occur efficiently within minutes on exposed positions in flexible sequences. The freshly liberated thiol group does not require treatment with reducing agents. We demonstrate the potential of this approach for protein modification with conceptually new schemes for regioselective dual labeling, thiol bioconjugation in presence of a preserved disulfide bond and formation of a novel intramolecular thioether crosslink.

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The real part of conductivity was measured for a number of salt solutions in different polar (nonaqueous) solvents over a broad frequency range up to 72 GHz at 20°C. The slope of the double log representation of conductivity versus frequency decreases with increasing salt concentration and solution viscosity. An extrapolation to infinite viscosity yields slope values similar to those found with vitreous systems which are claimed to exhibit “universal” behaviour.

The thiol group of the cysteine side chain is arguably the most versatile chemical handle in proteins. To expand the scope of established and commercially available thiol bioconjugation reagents, we genetically encoded a second such functional moiety in form of a latent thiol group that can be unmasked under mild physiological conditions. Phenylacetamidomethyl (Phacm) protected homocysteine (HcP) was incorporated and its latent thiol group unmasked on purified proteins using penicillin G acylase (PGA). The enzymatic deprotection depends on steric accessibility, but can occur efficiently within minutes on exposed positions in flexible sequences. The freshly liberated thiol group does not require treatment with reducing agents. We demonstrate the potential of this approach for protein modification with conceptually new schemes for regioselective dual labeling, thiol bioconjugation in presence of a preserved disulfide bond and formation of a novel intramolecular thioether crosslink.