Prof. Wen-Bin Zhang (College of Chemistry and Molecular Engineering, Peking University & Beijing Academy of Artificial Intelligence) and Dr. Jing Fang (College of Chemistry and Molecular Engineering, Peking University) are leading this study. A single domain protein catenane refers to two mechanically interlocking polypeptide rings that synergistically fold into a compact and integrated structure, which is extremely rare in nature. This design was achieved by rewiring the connectivity between secondary motifs to introduce artificial interlocking, and the synthesis could be easily achieved through a series of programmed, optimized post-translational processing events in cells without additional in vitro reactions.
The single-domain catenane, Katze-DHFR, was thoroughly characterized. Evidence from combined SDS-PAGE, SEC, LC-MS, IMS-MS, and proteolytic digestion experiments clearly proved its topology. The Katze-DHFR has improved anti-aggregation properties and has a TM that is 6°C higher than the linear control. Although the catalytic activity of Katze-DHFR is reduced due to its decreased substrate and cofactor affinity, it exhibits better thermal resilience than L-DHFR. Even after 10 minutes of incubation at 70°C, Katze-DHFR retained over 70% of its catalytic activity, while the linear control lost almost all of its activity. The research team believes that this method could be generally applicable to other single domain proteins, including those with DHFR-like or completely different folds. The availability of these single domain protein catenanes facilitates the elucidation of topological effects on structure-property relationships. The results also suggest that it is possible to map the current linear protein universe into single domain protein catenanes with well-preserved functions and additional advantages, opening up new frontiers for protein molecules. These topological proteins surpass the linear paradigm of natural protein molecules and are multi-chain, multi-dimensional molecules with functional advantages of topology, diverse design possibilities, and excellent evolvability. As a new class of protein molecules, they hold great potential for a wide range of applications, including but not limited to industrial enzymes, antibodies, cytokines, and biomaterials.
Fang, J., et al. (2023). A single-domain protein catenane of dihydrofolate reductase. National Science Review. doi.org/10.1093/nsr/nwad304.