Fig. 1: Cysteine scan for reversibly inhibitable SpyCatcher003 mutants.
A Model of the SpyTag-SpyCatcher reaction based on the crystal structure20. SpyCatcher (green) reacts with SpyTag (red), forming a covalent isopeptide bond between aspartate 7 in SpyTag and lysine 31 in SpyCatcher. B Heat map depicting the cysteine scan of SpyCatcher003 for reversible inhibition upon modification with Ellman’s reagent (DTNB). The first two rows show position and amino acid of SpyCatcher003, with reactive and catalytic residues in blue. The third row signifies amino acid conservation among SpyCatcher001, SpyCatcher002, and SpyCatcher003, using green for conserved and red for differing positions. The fourth row assesses reactivity of unmodified cysteine mutants with SpyTag within 90 min: green for fully reactive, yellow for slightly reduced reactivity, orange for strongly reduced reactivity, and red for unreactive. The fifth row evaluates reversible inhibition potential with Ellman’s reagent: green for yes, red for no, and white for not tested. C Reaction of a cysteine residue with Ellman’s reagent (DTNB) and reduction with TCEP.
