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<p>The non-covalent interactions of ubiquitin/ubiquitin-like proteins (Ub/Ubls) with other proteins play a pivotal role in regulating proteins recruitment and activity and ensuring proper protein modifications. However, capturing and identifying transient Ub/Ubl-mediated protein interactions in live cells remains a significant challenge. Genetically encoded cross-linking has emerged as a powerful tool to map such dynamic interactions. Although BprY, a cross-linkable unnatural amino acid (Uaa), has been incorporated into Ub/Ubls for mapping protein-protein interactions (PPIs), its cytotoxicity restricts its practical utility in live cells. Alkyne is non-toxic to cells, and the nucleophilic thiol-alkyne addition reaction facilitates the formation of a covalent thiovinyl bond. This bond can be used for the covalent capture of dynamic and instantaneous PPIs. In this work, we site-specifically incorporate alkyne-based Uaas, 4-propargyloxy-<i>L</i>-phenylalanine (pPR) or enrichable 4-propynyloxy-<i>L</i>-phenylalanine (epPR) into Ub/Ubls. This enables capture and identification of their binding partners in live cells. To extend this technology to polyubiquitin chains, we harness SortaseA-mediated transpeptidation along with Uaas, <i>N</i><sup>6</sup>-((2-azidoacetyl)glycyl)-<i>L</i>-lysine (AzGGK), pPR, and epPR to generate cross-linkable branched diubiquitin (diUb). Furthermore, we apply the rare codon recoding (RCR) system to multisite incorporate distinct types of Uaas, epPR, and enrichable fluorosulfate-<i>L</i>-tyrosine (eFSY) into ubiquitin-fold modifier 1 (UFM1) to enhance the efficiency of capturing interacting proteins. </p>

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Genetically Encoded Alkyne-Based Cross-linkable Probes

  • 期刊论文

  • 发表日期：

    2026年3月27日

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  • Wei Sun,
  • Yingchun Ni,
  • Xin Shu,
  • Dandan Liu,
  • Haifan Wu,
  • Shixian Lin,
  • Rong Zhou,
  • Bing Yang

摘要

The non-covalent interactions of ubiquitin/ubiquitin-like proteins (Ub/Ubls) with other proteins play a pivotal role in regulating proteins recruitment and activity and ensuring proper protein modifications. However, capturing and identifying transient Ub/Ubl-mediated protein interactions in live cells remains a significant challenge. Genetically encoded cross-linking has emerged as a powerful tool to map such dynamic interactions. Although BprY, a cross-linkable unnatural amino acid (Uaa), has been incorporated into Ub/Ubls for mapping protein-protein interactions (PPIs), its cytotoxicity restricts its practical utility in live cells. Alkyne is non-toxic to cells, and the nucleophilic thiol-alkyne addition reaction facilitates the formation of a covalent thiovinyl bond. This bond can be used for the covalent capture of dynamic and instantaneous PPIs. In this work, we site-specifically incorporate alkyne-based Uaas, 4-propargyloxy-L-phenylalanine (pPR) or enrichable 4-propynyloxy-L-phenylalanine (epPR) into Ub/Ubls. This enables capture and identification of their binding partners in live cells. To extend this technology to polyubiquitin chains, we harness SortaseA-mediated transpeptidation along with Uaas, N6-((2-azidoacetyl)glycyl)-L-lysine (AzGGK), pPR, and epPR to generate cross-linkable branched diubiquitin (diUb). Furthermore, we apply the rare codon recoding (RCR) system to multisite incorporate distinct types of Uaas, epPR, and enrichable fluorosulfate-L-tyrosine (eFSY) into ubiquitin-fold modifier 1 (UFM1) to enhance the efficiency of capturing interacting proteins.