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oai:figshare.com:article/34037742 2026-10-01T02:03:35Z category_1 category_4 category_915 category_7 category_8 category_13 category_14 category_21 category_24 category_61 category_132 category_134 category_135 portal_63 item_type_6 month_year_10_2026

Directed Evolution of a Protein Catenane Reveals How Noncanonical Topology Modulates Sequence–Property Relationships Jing Fang (170284) Zhaorong Chen (19710364) Yajie Liu (2590417) Wen-Bin Zhang (1298145) Biophysics Biochemistry Physical Sciences not elsewhere classified Medicine Microbiology Genetics Molecular Biology Biotechnology Evolutionary Biology Developmental Biology Infectious Diseases Virology Computational Biology yet soluble yield reverting evolved catenanes protein catenane reveals permitting substantial transferability enhanced substrate binding domain protein catenanes combined homologue expansion simultaneously improved stability linear counterparts transferred compare matched linear alternative topological isoforms increasing thermal stability thermal stability linear proteins catenated isoforms using single type dhfr tractable system sequence variation sequence background results demonstrate phenotypic effects largely preserving functional gains directed evolution dhfr homologues compact structure cat </ >- dhfr 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 (<i>cat</i>-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 <i>cat</i>-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. 2026-09-30T00:00:00Z Text Journal contribution 10.1021/jacs.6c11777.s001 https://figshare.com/articles/journal_contribution/Directed_Evolution_of_a_Protein_Catenane_Reveals_How_Noncanonical_Topology_Modulates_Sequence_Property_Relationships/34037742 CC BY-NC 4.0