ArticleBioorganic & medicinal chemistry2013
Nonenzymatic assembly of branched polyubiquitin chains for structural and biochemical studies.
Article in Bioorganic & medicinal chemistry, 2013. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 25 papers.
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Who cites it
25 citing papers in PubMed.
- AmideBiomolecular NMR assignments · 2026Article
- Thioether-mediated protein ubiquitination in constructing affinity- and activity-based ubiquitinated protein probes.Nature protocols · 2025Review
- Investigating the Interaction Profile of Unconjugated Ubiquitin: Chemical Biology and Affinity Enrichment Mass Spectrometric Approaches.Chembiochem : a European journal of chemical biology · 2025Review
- Emerging tools and methods to study cell signalling mediated by branched ubiquitin chains.Biochemical Society transactions · 2025Review
- Convergent Assembly of Homo- and Heterotypic Ubiquitin Chains from Functionalized, Expressed Monomers via Thiol-Ene Chemistry.Angewandte Chemie (International ed. in English) · 2025Article
- Genetic Code Expansion Approaches to Decipher the Ubiquitin Code.Chemical reviews · 2024Review
- Biocompatible Lysine Protecting Groups for the Chemoenzymatic Synthesis of K48/K63 Heterotypic and Branched Ubiquitin Chains.ACS central science · 2023Article
- Triubiquitin Probes for Identification of Reader and Eraser Proteins of Branched Polyubiquitin Chains.ACS chemical biology · 2023Article
- Functional analysis of protein post-translational modifications using genetic codon expansion.Protein science : a publication of the Protein Society · 2023Review
- A modular toolbox to generate complex polymeric ubiquitin architectures using orthogonal sortase enzymes.Nature communications · 2021Article
- Chemical methods for protein site-specific ubiquitination.RSC chemical biology · 2021Review
- Branched Ubiquitination: Detection Methods, Biological Functions and Chemical Synthesis.Molecules (Basel, Switzerland) · 2020Review
- Artificially Linked Ubiquitin Dimers Characterised Structurally and Dynamically by NMR Spectroscopy.Chembiochem : a European journal of chemical biology · 2019Article
- Analysis of the topology of ubiquitin chains.Methods in enzymology · 2019Article
- A Highly Efficient Synthesis of Polyubiquitin Chains.Advanced science (Weinheim, Baden-Wurttemberg, Germany) · 2018Article
- Review
- Subunit-Specific Labeling of Ubiquitin Chains by Using Sortase: Insights into the Selectivity of Deubiquitinases.Chembiochem : a European journal of chemical biology · 2016Article
- Chemical ubiquitination for decrypting a cellular code.The Biochemical journal · 2016Review
- Preparing to read the ubiquitin code: characterization of ubiquitin trimers by top-down mass spectrometry.Journal of mass spectrometry : JMS · 2016Article
- A Rapid and Versatile Method for Generating Proteins with Defined Ubiquitin Chains.Biochemistry · 2016Article
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5 authors.
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Abstract
Polymeric chains of a small protein ubiquitin are involved in regulation of nearly all vital processes in eukaryotic cells. Elucidating the signaling properties of polyubiquitin requires the ability to make these chains in vitro. In recent years, chemical and chemical-biology tools have been developed that produce fully natural isopeptide-linked polyUb chains with no need for linkage-specific ubiquitin-conjugating enzymes. These methods produced unbranched chains (in which no more than one lysine per ubiquitin is conjugated to another ubiquitin). Here we report a nonenzymatic method for the assembly of fully natural isopeptide-linked branched polyubiquitin chains. This method is based on the use of mutually orthogonal removable protecting groups (e.g., Boc- and Alloc-) on lysines combined with an Ag-catalyzed condensation reaction between a C-terminal thioester on one ubiquitin and a specific ε-amine on another ubiquitin, and involves genetic incorporation of more than one Lys(Boc) at the desired linkage positions in the ubiquitin sequence. We demonstrate our method by making a fully natural branched tri-ubiquitin containing isopeptide linkages via Lys11 and Lys33, and a (15)N-enriched proximal ubiquitin, which enabled monomer-specific structural and dynamical studies by NMR. Furthermore, we assayed disassembly of branched and unbranched tri-ubiquitins as well as control di-ubiquitins by the yeast proteasome-associated deubiquitinase Ubp6. Our results show that Ubp6 can recognize and disassemble a branched polyubiquitin, wherein cleavage preferences for individual linkages are retained. Our spectroscopic and functional data suggest that, at least for the chains studied here, the isopeptide linkages are effectively independent of each other. Together with our method for nonenzymatic assembly of unbranched polyubiquitin, these developments now provide tools for making fully natural polyubiquitin chains of essentially any type of linkage and length.
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