Enhancing protein stability and robustness with interlocking rings

This study was led by 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). A single-domain protein catenan refers to two mechanically interconnected polypeptide rings that fold synergistically 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 entanglement, and synthesis was easily achieved through a series of programmed enhanced post-translational processing events in cells without additional in vitro reactions.

The catenane of a domain Cat-DHFR was thoroughly characterized. Evidence from combined SDS-PAGE, SEC, LC-MS, IMS-MS and proteolytic digestion experiments clearly demonstrated their topology. The Cat-DHFR exhibits enhanced anti-aggregation properties and has a T_M i.e. 6 °C higher than the linear control. Although the catalytic activity of Cat– DHFR is reduced due to its reduced affinity for substrate and cofactor, it has better thermal stability than large-DHFR. Even after incubation at 70 °C for 10 min, Cat-DHFR retained over 70% of the catalytic activity, while the linear control lost almost all activity. The research team anticipates that this method could be generally applied to other single-domain proteins, including those with folds similar to DHFR or with completely different folds. The availability of these single-domain protein catenans facilitates the elucidation of topological effects on structure-property relationships. The results further suggest that it is possible to map the current linear protein universe into single-domain protein catenas with well-conserved functions and additional benefits, opening up new territory for protein molecules. Going beyond the linear paradigm of natural protein molecules, these topological proteins are multi-chain, multi-dimensional molecules with functional benefits of topology, rich design potential, and extraordinary evolvability. As a new class of protein molecules, they have great potential for a wide range of applications, including but not limited to industrial enzymes, antibodies, cytokines, and biomaterials.