Design and biosynthesis of catenated dihydrofolate reductase (DHFR) (IMAGE)
Caption
(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. (c) Structure prediction (https://robetta.bakerlab.org/) of l-DHFR. (d) The Gaussian linking number (GLN) matrix of wt-DHFR. It comprises GLN values between all neighboring residue pairs within the same chain. The sum of all the cells within the boxed sub-matrix corresponds to the GLN value between the two subchains, which provides a quantitative metric of the extent of their entanglement.
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