Determination of binding free energy shifts in protein complexes induced by single and double amino acid mutations using molecular dynamics simulations

Binding free energy between protein molecules strongly influences protein functions. In particular, shifts in binding free energy caused by amino acid mutations alter the conformational stability of two protein complexes, potentially resulting in functional changes. Predicting these changes in binding free energy due to amino acid mutations remains a considerable challenge. In this study, we propose a simple procedure to calculate the binding free energy shifts in protein complexes resulting from amino acid mutations. We applied this method to the Barnase–Barstar complex, performing molecular dynamics simulations combined with alchemical free energy calculations. Our analysis included three single mutants of Barnase, seven single mutants of Barstar, and three double mutants. The predicted binding free energy shifts were generally consistent with the experimental results, with the exception of two cases. We showed that our method is effective for predicting binding free energy changes when the amino acid mutations do not induce large-scale conformational changes in the protein complex.