Directed Evolution of a Protein Catenane Reveals How Noncanonical Topology Modulates Sequence–Property Relationships

Abstract

Single-domain protein catenanes, in which two mechanically interlocked polypeptide rings fold into a compact structure, provide a tractable system for probing how chemical topology reshapes protein fitness. Using single-domain catenated dihydrofolate reductase (cat-DHFR) as a model, we combined homologue expansion, directed evolution, and topology reversion to compare matched linear and catenated isoforms. Catenation proved broadly compatible with DHFR homologues, yet soluble yield and catalytic activity remained strongly sequence dependent. Directed evolution substantially improved the catalytic activity of cat-DHFR through enhanced substrate binding and turnover, while largely preserving or increasing thermal stability. Strikingly, reverting evolved catenanes to their linear counterparts transferred these functional gains: all reverted linear isoforms exhibited higher catalytic activity and thermal stability than wild-type DHFR. Across matched topological pairs, catenation generally increased thermal stability, whereas catalytic activity depended more strongly on sequence background and topology. These results demonstrate that chemical topology can modulate the phenotypic effects of sequence variation, while permitting substantial transferability of beneficial mutations across topological contexts. Protein catenanes may therefore serve not only as alternative topological isoforms, but also as evolutionary intermediates en route to linear proteins with simultaneously improved stability and activity.