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. 2019 Dec 10;116(52):26523–26533. doi: 10.1073/pnas.1909653116

Fig. 1.

Fig. 1.

Covalent peptide–protein reaction accelerated by 003 pair. (A) Spontaneous isopeptide bond formation in Spy CnaB2 domain and engineering to give SpyTag and SpyCatcher. The reactive Lys31 and Asp117 are shown in stick format based on PDB 2X5P and 4MLI. (B) Location of SpyCatcher003 mutations. Mutations in SpyCatcher002 to give SpyCatcher003 are marked in green in stick format. The reactive Lys and Asp are shown in spacefill. SpyTag is marked in red. S49 was mutated to Cys in certain constructs for dye attachment. Based on PDB 2X5P and 4MLI. (C) Reaction rate at 10 nM. The 10 nM SpyTag-sfGFP variants were incubated for the indicated time at 25 °C with 10 nM SpyCatcher variants. (D) Reaction rate with each partner at 100 nM, performed as in C. (E) Reaction rate with each partner at 10 µM, performed as in C. Data are mean ± 1 SD, n = 3; some error bars are too small to be visible.