Histone ubiquitination affects the structure and function of nucleosomes through tightly regulated dynamic reversible processes. The efficient preparation of ubiquitinated histones and their analogs is important for biochemical and biophysical studies on histone ubiquitination. Here, we report the CAACU (cysteine-aminoethylation assisted chemical ubiquitination) strategy for the efficient synthesis of ubiquitinated histone analogs. The key step in the CAACU strategy is the installation of an N-alkylated 2-bromoethylamine derivative into a recombinant histone through cysteine aminoethylation, followed by native chemical ligation assisted by Seitz’s auxiliary to produce mono- and diubiquitin (Ub) and small ubiquitin-like modifier (SUMO) modified histone analogs. This approach enables the rapid production of modified histones from recombinant proteins at about 1.5–6 mg/L expression. The thioether-containing isopeptide bonds in the products are chemically stable and bear only one atomic substitution in the structure, compared to their native counterparts. The ubiquitinated histone analogs prepared by CAACU can be readily reconstituted into nucleosomes and selectively recognized by relevant interacting proteins. The thioether-containing isopeptide bonds can also be recognized and hydrolyzed by deubiquitinases (DUBs). Cryo-electron microscopy (cryo-EM) of the nucleosome containing H2BKC34Ub indicated that the obtained CAACU histones were of good quality for structural studies. Collectively, this work exemplifies the utility of the CAACU strategy for the simple and efficient production of homogeneous ubiquitinated and SUMOylated histones for biochemical and biophysical studies.

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Activity-based E2 conjugating enzyme (E2)-ubiquitin (Ub) probes have recently emerged as effective tools for studying the molecular mechanism of E3 ligase (E3)-catalyzed ubiquitination. However,t he preparation of existing activitybased E2-Ub probes depends on recombination technology and bioconjugation chemistry,l imiting their structural diversity.H erein we describe an expedient total chemical synthesis of an E2 enzyme variant through ah ydrazide-based native chemical ligation, which enabled the construction of astructurally new activity-based E2-Ub probe to covalentlycapture the catalytic site of Cys-dependent E3s.C hemical cross-linking coupled with mass spectrometry (CXMS) demonstrated the utility of this new probe in structural analysis of the intermediates formed during Nedd4 and Parkin-mediated transthiolation. This study exemplifies the utility of chemical protein synthesis for the development of protein probes for biological studies.

Triazole‐based deubiquitylase (DUB)‐resistant ubiquitin (Ub) probes have recently emerged as effective tools for the discovery of Ub chain‐specific interactors in proteomic studies, but their structural diversity is limited. A new family of DUB‐resistant Ub probes is reported based on isopeptide‐N‐ethylated dimeric or polymeric Ub chains, which can be efficiently prepared by a one‐pot, ubiquitin‐activating enzyme (E1)‐catalyzed condensation reaction of recombinant Ub precursors to give various homotypic and even branched Ub probes at multi‐milligram scale. Proteomic studies using label‐free quantitative (LFQ) MS indicated that the isopeptide‐N‐ethylated Ub probes may complement the triazole‐based probes in the study of Ub interactome. Our study highlights the utility of modern protein synthetic chemistry to develop structurally and new families of tool molecules needed for proteomic studies.

Sortase A (SrtA)-mediated ligation, a popular method for protein labeling and semi-synthesis, is limited by its reversibility and dependence on the LPxTG motif, where "x" is any amino acid. Here, we report that SrtA can mediate the efficient and irreversible ligation of a protein/peptide containing a Cterminal thioester with another protein/peptide bearing an N-terminal Gly, with broad tolerance for a wide variety of LPxT-derived sequences. This strategy, the thioester-assisted SrtA-mediated ligation, enabled the expedient preparation of proteins bearing various N-or C-terminal labels, including post-translationally modified proteins such as the Ser139-phosphorylated histone H2AX and Lys9-methylated histone H3, with less dependence on the LPxTG motif. Our study validates the chemical modification of substrates as an effective means of augmenting the synthetic capability of existing enzymatic methods.

The chemical synthesis of homogeneously modified histones is a powerful approach to quantitatively decipher how post-translational modifications (PTMs) modulate epigenetic events. Herein, we describe the expedient syntheses of a selection of phosphorylated and ubiquitinated H2AX proteins in a strategy integrating expressed protein hydrazinolysis and auxiliary-mediated protein ligation. These modified H2AX proteins were then used to discover that although H2AXS139 phosphorylation can enhance the binding of the DNA damage repair factor 53BP1 to either an unmodified nucleosome or that bearing a single H2AXK15ub or H4K20me2 modification, it augments 53BP1’s binding only weakly to nucleosomes bearing both H2AXK15ub and H4K20me2. To better understand why such a trivalent additive effect is lacking, we solved the cryo-EM structure (3.38 Å) of the complex of 53BP1 with the H2AXK15ub/S139ph_H4K20me2 nucleosome, which showed that H2AXS139 phosphorylation distorts the interaction interface between ubiquitin and 53BP1’s UDR motif. Our study revealed that there is redundancy in the interplay of multiple histone PTMs, which may be useful for controlling the dynamic distribution of effector proteins onto nucleosomes bearing different histone variants and PTMs in a time-dependent fashion, through specific cellular biochemical events.

Triazole‐based deubiquitylase (DUB)‐resistant ubiquitin (Ub) probes have recently emerged as effective tools for the discovery of Ub chain‐specific interactors in proteomic studies, but their structural diversity is limited. A new family of DUB‐resistant Ub probes is reported based on isopeptide‐N‐ethylated dimeric or polymeric Ub chains, which can be efficiently prepared by a one‐pot, ubiquitin‐activating enzyme (E1)‐catalyzed condensation reaction of recombinant Ub precursors to give various homotypic and even branched Ub probes at multi‐milligram scale. Proteomic studies using label‐free quantitative (LFQ) MS indicated that the isopeptide‐N‐ethylated Ub probes may complement the triazole‐based probes in the study of Ub interactome. Our study highlights the utility of modern protein synthetic chemistry to develop structurally and new families of tool molecules needed for proteomic studies.

Bioorthogonal reactions have emerged as valuable tools for site-specific protein labeling and modification in vitro and in vivo. Hydrazone and oxime ligation has recently attracted considerable attention for wide applications in the conjugation of biomolecules. However, this kind of reaction has suffered from slow kinetics under physiological conditions and toxicity or complications of the reaction system due to catalysts. In this work we have developed an electron-deficient benzaldehyde reagent, which can be easily equipped with various types of bio-functional molecules for catalyst-free hydrazone ligation. The reagent can be equipped with not only small molecules such as fluorescence dyes or drugs, but also macromolecules like PEG. These can be precisely ligated to the C-terminus of proteins by an efficient hydrazone reaction at neutral pH and room temperature. The new reagent based catalyst-free hydrazone ligation provides a practical approach for the site specific modification of proteins.