Abstract：
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.
摘要：
读者的身份识别，一类重要的识别特定位点修饰残基的蛋白质，对于揭示翻译后修饰的生物学作用至关重要。光反应性交联剂是研究读者的有力工具。然而,现有方法通常采用具有综合挑战性的光反应弹头，以及它们在辐照时产生的高能中间体，如氮烯和卡宾，可能导致实质性的非特异性交联。在这里，我们将二甲基锍报告为甲基赖氨酸模拟物，该模拟物与特定的读取器结合，随后在紫外线照射下通过单电子转移与结合袋内的保守色氨酸交联。交联依赖于锍和吲哚之间的蛋白质模板σ-π电子供体-受体相互作用，确保色氨酸在活性位点的优异位点选择性和与其他甲基赖氨酸读取器的正交性。这种方法可以提高从复杂细胞样品中发现甲基赖氨酸读取器的程度。此外，这种光交联策略可以扩展到开发其他类型的微环境依赖性缀合位点特异性色氨酸.
