A single-domain green fluorescent protein catenane.

Natural proteins exhibit rich structural diversity based on the folds of an invariably linear chain. Macromolecular catenanes that cooperatively fold into a single domain do not belong to the current protein universe, and their design and synthesis open new territories in chemistry. Here, we report the design, synthesis, and properties of a single-domain green fluorescent protein catenane via rewiring the connectivity of GFP's secondary motifs. The synthesis could be achieved in two steps via a pseudorotaxane intermediate or directly via expression in cellulo. Various proteins-of-interest may be inserted at the loop regions to give fusion protein catenanes where the two subunits exhibit enhanced thermal resilience, thermal stability, and mechanical stability due to strong conformational coupling. The strategy can be applied to other proteins with similar fold, giving rise to a family of single-domain fluorescent proteins. The results imply that there may be multiple protein topological variants with desirable functional traits beyond their corresponding linear protein counterparts, which are now made accessible and fully open for exploration.