Xu Lianjie, Tian Xibao, Zhang Wen-Bin
Beijing National Laboratory for Molecular Sciences, Key Laboratory of Polymer Chemistry & Physics of Ministry of Education, Center for Soft Matter Science and Engineering, College of Chemistry and Molecular Engineering, Peking University Beijing 100871 P. R. China
AI for Science (AI4S)-Preferred Program, Shenzhen Graduate School, Peking University Shenzhen 518055 P. R. China.
Chem Sci. 2025 Aug 8. doi: 10.1039/d5sc03953c.
Synthesis of nontrivial protein topologies calls for genetically encoded protein entangling motifs, especially those of heterogeneous nature, to achieve structural complexity and functional relevance. Herein, we report the systematic discovery of heterodimeric entangling motifs using criteria like Gauss linking number, buried surface area and terminal distances. These motifs were analyzed to reveal their formation mechanisms (, precursor cleavage, synergistic folding and segment piercing/wrapping) and biological significance (, stability enhancement crucial for executing functions like regulation and catalysis). Six premium motifs were selected for experimental validation. Upon ring closure mediated by orthogonal split inteins, all six motifs led to protein hetero[2]catenanes with varying efficiency, providing versatile templates for making mechanically interlocked protein conjugates, such as Förster resonance energy transfer pairs and bispecific binders. The study not only helps untangle the influence of chain entanglements on protein properties but also provides a modular platform to enrich the toolbox of protein topology engineering.
非平凡蛋白质拓扑结构的合成需要遗传编码的蛋白质缠结基序，尤其是那些具有异质性的基序，以实现结构复杂性和功能相关性。在此，我们报告了使用高斯链接数、埋藏表面积和末端距离等标准对异二聚体缠结基序的系统发现。对这些基序进行了分析，以揭示它们的形成机制（即前体切割、协同折叠和片段穿透/包裹）和生物学意义（即增强稳定性，这对于执行调节和催化等功能至关重要）。选择了六个优质基序进行实验验证。在由正交分裂内含肽介导的环化作用下，所有六个基序都以不同效率产生了蛋白质异[2]连环体，为制备机械互锁的蛋白质缀合物提供了通用模板，如荧光共振能量转移对和双特异性结合剂。该研究不仅有助于理清链缠结对蛋白质性质的影响，还提供了一个模块化平台来丰富蛋白质拓扑工程的工具箱。
