Computational Design and Cellular Synthesis of Two Protein Topological Isoforms: Solomon Link vs Three-Twist Knot

Topology has emerged as a unique dimension in protein engineering, drawing attentions to the pursuit of nontrivial protein topologies due to their functional benefits like enhanced stability and rich dynamics. However, the complexity and diversity of artificial topological proteins remain rather limited to date. Herein, we report computational design and cellular synthesis of a pair of topological isoforms based on the symmetric assembly of orthogonal entangling motifs. Two orthogonal entangling motifs with C2 symmetry, i.e., p53dim and HP0242, are fused in different ways to direct the formation of multiple crossings to generate a protein Solomon link and a protein three-twist knot upon cyclization. The fusion order and linker lengths are carefully chosen to narrow the range of possible protein topologies down to the target ones. Selective syntheses of designed protein topologies in cells are evidenced by experimental characterizations including sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), size exclusion chromatography (SEC), and liquid chromatography-mass spectrometry (LC-MS). Notably, both Solomon link and three-twist knot exhibit higher compactness and stability than their corresponding topological controls (such as catenane, trefoil knot, and linear forms) as evidenced by their advantageous properties in thermal stability and resistance towards chemical denaturation. As a rational and powerful method to create diverse mechanically interlocked topological proteins from orthogonal entangling motifs, this approach may be extended to build even more complex topologies like protein chain-mails or weaved protein frameworks.