Following the docking simulations, we produced recombinant HSA (rHSA) with the Cys476-to-Gly mutation using site-directed mutagenesis (SDM). Experimental validation confirmed that the C476G mutation led to an expansion of the protein cavity, which facilitated easier access of CO-1080 to the binding site. The mutation also exposed free –SH groups that accelerated the covalent binding reaction between CO-1080 and rHSA, particularly under mild conditions at room temperature. These findings confirmed the success of the C476G mutation in improving both non-covalent and covalent binding interactions.
