A single-domain protein catenane refers to two mechanically interlocked polypeptide rings that fold synergistically into a compact and integrated structure, which is extremely rare in nature. Herein, we report a single-domain protein catenane of dihydrofolate reductase (cat-DHFR). The design was achieved by rewiring the connectivity between secondary motifs to introduce artificial entanglement and the synthesis was readily accomplished by a series of programmed streamlined post-translational processing events in cells without any additional in vitro reactions. The target molecule contains few exogenous motifs and has been thoroughly characterized by combined techniques of LC-MS, SDS-PAGE, protease cleavage experiment, and ion mobility mass spectrometry. Compared to the linear control, cat-DHFR retains the catalytic capability and exhibits enhanced stability against thermal or chemical denaturation due to conformational restriction. The results suggest that linear proteins may be converted into concatenated single-domain counterparts with almost identical chemical composition, well-preserved function, and elevated stability, which represents an entirely new horizon in protein science.

A single-domain protein catenane refers to two mechanically interlocked polypeptide rings that fold synergistically into a compact and integrated structure, which is extremely rare in nature. Herein, we report a single-domain protein catenane of dihydrofolate reductase (cat-DHFR). The design was achieved by rewiring the connectivity between secondary motifs to introduce artificial entanglement and the synthesis was accomplished by a series of programmed streamlined post-translational processing events in cells. The target molecule contains few exogenous motifs and has been thoroughly characterized by combined techniques of LC-MS, SDS-PAGE, protease cleavage experiment, and ion mobility mass spectrometry. Compared to the linear control, cat-DHFR retains the catalytic capability and exhibits enhanced stability against thermal or chemical denaturation due to conformational restriction. The results suggest that linear proteins may be converted into concatenated single-domain counterparts with almost identical chemical composition, well-preserved function, and elevated stability, which represents an entirely new horizon in protein science.