Ubiquitin Chains Bearing Genetically Encoded Photo-Cross-Linkers Enable Efficient Covalent Capture of (Poly)ubiquitin-Binding Domains

Ubiquitin-mediated signaling pathways regulate essentially every aspect of cell biology in eukaryotes. Ubiquitin receptors typically contain ubiquitin-binding domains (UBDs) that have the ability to recognize monomeric ubiquitin (Ub) and polymeric Ub (polyUb) chains. However, how signaling specifici...

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Bibliographic Details
Published in:Biochemistry (Easton) 2019-02, Vol.58 (7), p.883-886
Main Authors: Braxton, Courtney N, Quartner, Evan, Pawloski, Westley, Fushman, David, Cropp, T. Ashton
Format: Article
Language:English
Subjects:
Benzophenones - chemistry
Binding Sites
Carrier Proteins - metabolism
Cross-Linking Reagents - chemistry
DNA-Binding Proteins
Histone Chaperones
Mutation
Nuclear Proteins - metabolism
Phenylalanine - analogs & derivatives
Phenylalanine - chemistry
Phenylalanine - genetics
Polyubiquitin - metabolism
Proteasome Endopeptidase Complex - metabolism
Protein Domains
RNA, Transfer, Tyr
Saccharomyces cerevisiae Proteins - metabolism
Ubiquitin - chemistry
Ubiquitin - genetics
Ubiquitin - metabolism
Ubiquitin-Activating Enzymes - metabolism
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Summary:Ubiquitin-mediated signaling pathways regulate essentially every aspect of cell biology in eukaryotes. Ubiquitin receptors typically contain ubiquitin-binding domains (UBDs) that have the ability to recognize monomeric ubiquitin (Ub) and polymeric Ub (polyUb) chains. However, how signaling specificity is achieved remains poorly understood, and many of the UBDs that selectively recognize polyUb chains of particular linkages still need to be identified and characterized. Here we report the incorporation of a genetically encoded photo-cross-linker, p-benzoyl-l-phenylalanine (Bpa), into recombinant Ub and enzymatically synthesized polyUb chains. This allows photo-cross-linking (covalent bond formation) of monoUb and K48- and K63-linked diUb chains to UBDs. This approach provides a framework for understanding Ub cellular signaling through the capture and identification of (poly)­Ub-binding proteins.
ISSN:0006-2960
1520-4995
DOI:10.1021/acs.biochem.8b01089