A Single-domain Protein Catenane of Dihydrofolate Reductase

Abstract

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. Here, we report a single-domain protein catenane of dihydrofolate reductase (cat-DHFR). This design was achieved by rewiring the connectivity between secondary motifs to introduce artificial entanglement, and synthesis was readily accomplished through a series of programmed streamlined post-translational processing events in cells without any additional in vitro reactions. The target molecule contained few exogenous motifs and was thoroughly characterized using a combination of LC-MS, SDS-PAGE, protease cleavage experiments, and ion mobility mass spectrometry. Compared with the linear control, cat-DHFR retained its catalytic capability and exhibited enhanced stability against thermal or chemical denaturation due to conformational restriction. These results suggest that linear proteins may be converted into their concatenated single-domain counterparts with almost identical chemical compositions, well-preserved functions, and elevated stabilities, representing an entirely new horizon in protein science.