The team led by Prof. Wen-Bin Zhang at the College of Chemistry and Molecular Engineering, Peking University designed and synthesized a single-domain protein catenane of dihydrofolate reductase (DHFR) and demonstrated the effects of catenation on protein properties. The research results were published online by National Science Review on November 29, 2023 (https://doi.org/10.1093/nsr/nwad304), with the title “A Single-domain Protein Catenane of Dihydrofolate Reductase”.
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. 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 (Fig. 1).
The catenane was thoroughly characterized by combined techniques of SDS–PAGE, SEC, LC–MS, IMS–MS, and proteolytic digestion to unambiguously prove its topology. The cat-DHFR exhibits enhanced anti-aggregation properties and a Tm 6 °C higher than the linear control (l-DHFR) (Fig. 2). Although the catalytic activity of cat-DHFR is reduced owing to its decreased affinity toward the substrate and cofactor, it has better thermal resilience than l-DHFR. Even after incubation at 70 °C for 10 min, over 70% of the catalytic activity was still retained, while the linear control lost almost all activity (Fig. 2). The research team anticipates that this method could also be generally applicable to other single-domain proteins, including those with folds similar to DHFR or with completely different folds. The availability of these single-domain protein catenanes facilitates the elucidation of topological effects on structure–property relationships. The results show that it is possible to construct a single-domain protein catenane from a linear protein precursor with well-preserved functions and additional benefits, opening up new territory for protein molecules. Surpassing the linear paradigm of natural protein molecules, these topological proteins are intrinsically multi-chain, multi-dimensional molecules that possess more design space and better evolvability, in addition to the functional benefits of topology. As a new class of protein molecules, they hold great potential for a broad range of applications, including, but not limited to, industrial enzymes, antibodies, cytokines, and biomaterials.
Fig. 1 Design and biosynthesis of catenated dihydrofolate reductase (DHFR).
(a) Protein topological diagram of l-DHFR (left) and cat-DHFR (middle), and the retrosynthetic analysis of cat-DHFR (right). Numbers 1–8 and letters A–D represent the β-sheet and α-helix, respectively, from N- to C- termini in consecutive order. The split site (residues 88 and 89) is located at the loop region between α-helix-C and β-sheet-5. The highlighted lines are the linkers generated when forming the cat-DHFR. The star denotes the possible position for ring I closure (at the same and opposite sides) and split-intein insertion. L2 is the linker newly introduced to ring II of cat-DHFR. (b) Scheme of the cat-DHFR biosynthesis process using programmed post-translation processing events. DHFR1 is circularly permutated, and the corresponding sequences are denoted by DHFR1-1 and DHFR1-2. The TEV recognition site and a GG linker were inserted into ring I. The His-tag and a variable linker (together, they are L2) were inserted into ring II.
Fig.2 Effects of catenation on DHFR properties.
(a) Schematic diagram of DHFR topology traversing. (b) Differential scanning calorimetry characterization and melting temperature (Tm) values for l- and cat-DHFR. (c) Anti-aggregation properties of l- and cat-DHFR. The samples (20 μM) were incubated at 85 °C for 1–6 h, and the supernatant was monitored by SDS–PAGE. (d) Percentage of catalytic activity preserved for l- and cat-DHFR after incubation at 50 °C, 55 °C, 60 °C, 65 °C, and 70 °C for 10 min.
Jing Fang, the Boya Postdoctoral Fellow at the College of Chemistry and Molecular Engineering, Peking University, is the first author of this paper. Prof. Wen-Bin Zhang from the College of Chemistry and Molecular Engineering, Peking University, is the corresponding author. The team of Prof. Jongcheol Seo of Pohang University of Science and Technology provided important help in the characterization of the topological structure of catenated dihydrofolate reductase. This work was supported by the National Key Research and Development Program, the National Natural Science Foundation of China, the Beijing National Laboratory for Molecular Sciences, and the National Research Foundation of Korea.
Original link for the paper: https://doi.org/10.1093/nsr/nwad304.
