Department of Chemistry, Zhejiang University, Hangzhou, China.
Department of Chemistry, School of Science, Westlake University, Hangzhou, China.
Nat Chem. 2024 Aug;16(8):1267-1277. doi: 10.1038/s41557-024-01577-y. Epub 2024 Jul 30.
The identification of readers, an important class of proteins that recognize modified residues at specific sites, is essential to uncover the biological roles of post-translational modifications. Photoreactive crosslinkers are powerful tools for investigating readers. However, existing methods usually employ synthetically challenging photoreactive warheads, and their high-energy intermediates generated upon irradiation, such as nitrene and carbene, may cause substantial non-specific crosslinking. Here we report dimethylsulfonium as a methyllysine mimic that binds to specific readers and subsequently crosslinks to a conserved tryptophan inside the binding pocket through single-electron transfer under ultraviolet irradiation. The crosslinking relies on a protein-templated σ-π electron donor-acceptor interaction between sulfonium and indole, ensuring excellent site selectivity for tryptophan in the active site and orthogonality to other methyllysine readers. This method could escalate the discovery of methyllysine readers from complex cell samples. Furthermore, this photo crosslinking strategy could be extended to develop other types of microenvironment-dependent conjugations to site-specific tryptophan.
读者蛋白是一类能够识别特定位点上修饰残基的重要蛋白质，对揭示翻译后修饰的生物学功能至关重要。光反应交联剂是研究读者蛋白的有力工具。然而，现有的方法通常采用具有挑战性的合成光反应弹头，其在辐照时产生的高能中间体，如氮宾和卡宾，可能会导致大量的非特异性交联。在这里，我们报告二甲硫鎓作为一种赖氨酸甲酯类似物，它可以与特定的读者结合，然后在紫外辐照下通过单电子转移与结合口袋内的保守色氨酸发生交联。这种交联依赖于硫鎓和吲哚之间的蛋白质模板化的 σ-π 电子供体-受体相互作用，确保了活性位点中色氨酸的优异的位点选择性和与其他赖氨酸甲酯读者的正交性。这种方法可以从复杂的细胞样本中发现更多的赖氨酸甲酯读者蛋白。此外，这种光交联策略可以扩展到开发其他类型的依赖微环境的对特定色氨酸的偶联反应。
