To investigate the binding kinetics PB-3 with PLY-C428A the biolayer-interferometry assay was employed. For this, both His-tagged wild-type PLY and His-tagged PLY-C428A were immobilized on two separate Ni-NTA biosensors. The protein-coated biosensors were immersed in buffer with various concentrations (2, 4 and 8 µM) of PB-3 of PB-3 and binding was monitored over time. While association of PB-3 was observed to both protein sensors, binding to wildtype PLY was stronger compared to the mutant PLY-C428A (Figure ), which confirmed that presence of Cys428 in the protein enhanced the binding affinity of PB-3. A putative covalent binding mode of PB-3 with PLY was generated using molecular modeling and indicated the accessibility of the electrophilic double bond for Michael addition of C428 as well as binding of PB-3 through hydrophobic interactions with the amino acid residues of domain 4 with (Figure ).