Benchmarking Free Energy Calculations: Analysis of Single and Double Mutations Across Two Simulation Software...

Mutation analysis of single and double mutations of proteins including for the two widely studied proteins: Staphylococcus nuclease (S. nuclease) and T4 lysozyme, provide critical insights into their stability and fitness. Furthermore, such analysis facilitates a deeper understanding of the complex interplay between a protein's sequence, structural conformation, and functional output. Free Energy Perturbation (FEP) is an alchemical, all-atom molecular dynamics based computational approach that determines the free energy change ( $\Delta \Delta G$ ) from wild type to mutant states. Two widely adopted software platforms used for this purpose are Schrödinger and GROMACS. We have compared the results of FEP simulations for mutations using these platforms, employing the OPLS4 force field implemented in Schrӧdinger, and the Amber99SB-ILDN force field implemented in GROMACS for this work. For the 38 single mutants of S. nuclease, the Pearson r between the experimental and the calculated free energy change ( $\Delta \Delta G$ ) was 0.86 using Schrödinger and 0.87 using GROMACS. The reliability of the FEP method using the two software platforms with the specified force fields was further demonstrated by its performance on 24 single mutants of T4 lysozyme, yielding strong correlation between predicted and experimental $\Delta \Delta G$ values, with Pearson r of 0.80 for Schrödinger and 0.85 for GROMACS. Additionally, the computed folding free energy changes for 45 double mutations in S. nuclease ( $\Delta \Delta G_{WT}^{AB}$ ) using Schrödinger correlated well with both experimental measurements (Pearson r = 0.74) and previously reported GROMACS values (Pearson r = 0.71). Correspondingly, the nonadditivities( ${\delta }_{WT}^{AB}$ ) of the double mutations derived using Schrӧdinger for these 45 double mutants of S. nuclease were also found to be in good agreement with the experimental values (Pearson r = 0.79), as well as with the previously reported GROMACS results (Pearson r = 0.61). A good correlation was also observed between computed values from Schrödinger and GROMACS, with a Pearson r of 0.71 for double mutants and 0.61 for their nonadditivities. Collectively, these findings establish the efficiency, accuracy, and reliability of Schrödinger FEP+ and GROMACS in predicting mutation induced free energy changes in S. nuclease and T4 lysozyme. The integration of FEP based computational methodologies with experimental validation provides a framework for quantifying mutation induced changes in protein thermostability, which can be used as a tool for protein engineering and design.