The team led by Prof. Wen-Bin Zhang at the College of Chemistry and Molecular Engineering, Peking University, hasreported the firstdesign and synthesis of single-domain green fluorescent protein (GFP) catenanes via rewiring the connectivity of GFP's secondary motifs. The strategy can be applied to other proteins with similar folds, giving rise to a family of single-domain fluorescent proteins. It also offers a new scaffold for making fusion protein catenanes. The research results were published online in Nature Communications on June 13, 2023 (https://doi.org/10.1038/s41467-023-39233-7) with the title “A single-domain green fluorescent protein catenane”.
Topology concerns the connectivity and spatial relationship of a molecule, and is an important molecular attribute for macromolecules. Out of the vast majority of natural peptides or proteins, only a small fraction of them gains nontrivial topologies through post-translational processing, which is thought to bring in certain functional benefits, such as stabilization. In recent years, efforts have been made to construct artificial topological proteins using “reaction-assembly” synergy, but most of them are multi-domain which contain relics from entangling templates. Itrepresents a grand challenge to design and synthesize a single-domain protein catenane since most proteins are free of entanglement. Herein, the team led by Prof. Wen-Bin Zhang reported a versatile strategy for the design and synthesis of single-domain protein catenanes, using GFP as a model protein, through rewiring the connectivity between different secondary motifs to introduce entanglement within a single domain(Fig. 1).
Fig.1 Design of a single-domain GFP catenane through rewiring the connectivity between different secondary motifs.(a) Cartoon illustration, topology diagram, and gene encoding the original GFP (PDB: 2B3P). (b) Cartoon illustration, topology diagram, and gene encoding single-domain GFP catenane.
A two-step templated strategy via a pseudorotaxane intermediate (rtx-GFP) was developed. Through ringclosure in vitro, rtx-GFP was converted to [2]catenane (denoted as [2]cat-GFP) through intramolecular reaction and [3]catenane (denoted as [3]cat-GFP) through intermolecular reaction(Fig. 2a). The method also works for proteins with similar fold, giving rise to a family of single-domainprotein catenanes like cat-YPet, cat-mCherry, and cat-Wasabi. Moreover, it also provides a robust scaffold to make fusion catenanes with POIs inserted at the loop region (cat-GFP-POI)(Fig. 2b).
Fig. 2 (a) Two-step synthesis of GFP catenane via a pseudorotaxane intermediate.(b) Two-step synthesis offusion protein catenanes.
The effects of mechanical interlocking were then explored. In comparison to the linear and cyclic controls, cat-GFP(-POI) shows the best thermal resilience, with a sharp slope and nearly 100% recovery of fluorescence after being boiled(Fig. 3a). Due to conformational restriction, the embedded POIs exhibit improved thermal stability, thermal resilience, and mechanical stability(Fig. 3b). The results suggest that mechanical coupling between interlocked subunits within catenane topology could bring in significant changes in properties.
Fig. 3 (a) cat-GFP(-POI) shows the best thermal resilience. (b)Embedded POIs exhibit improved thermal stability, thermal resilience, and mechanical stability.
Zhiyu Qu, a Ph.D. candidate at Peking University is the first author of this paper. Prof. Wen-Bin Zhang from Peking University is the corresponding author. Jing Fang, Yu-Xiang Wang, Yibin Sun, Yajie Liu, and Wen-Hao Wu from Peking University also contribute to this work. This research was jointly supported by the National Natural Science Foundation of China, the National Key R&D Program of China, and Beijing NationalLaboratory for Molecular Sciences.
Original link for the paper: https://doi.org/10.1038/s41467-023-39233-7.
