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

Area of Science:

• Computational Biology and Bioinformatics
• Protein Engineering and Design
• Molecular Dynamics Simulations

Background:

• Protein stability and fitness are critically influenced by mutations.
• Understanding sequence-structure-function relationships is key to protein analysis.
• Free Energy Perturbation (FEP) is a computational method for quantifying mutation effects.

Purpose of the Study:

• To compare the accuracy and reliability of Schrödinger and GROMACS FEP simulations.
• To evaluate the prediction of mutation-induced free energy changes ($\Delta \Delta G$) for S. nuclease and T4 lysozyme.
• To assess the performance of FEP in predicting double mutation nonadditivities.

Main Methods:

• Utilized Free Energy Perturbation (FEP) simulations on single and double mutants.
• Employed Schrödinger with OPLS4 force field and GROMACS with Amber99SB-ILDN force field.
• Compared computed $\Delta \Delta G$ and nonadditivity values against experimental data.

Main Results:

• High correlations (Pearson r > 0.80) were observed between computed and experimental $\Delta \Delta G$ for single mutants of S. nuclease and T4 lysozyme using both platforms.
• Schrödinger and GROMACS showed good agreement with experimental data for double mutation free energy changes (Pearson r = 0.74) and nonadditivities (Pearson r = 0.79).
• Strong correlations were found between Schrödinger and GROMACS computed values for both single and double mutations.

Conclusions:

• Schrödinger FEP+ and GROMACS are efficient and reliable tools for predicting mutation-induced free energy changes.
• FEP simulations, when validated experimentally, provide a robust framework for quantifying changes in protein thermostability.
• This approach serves as a valuable tool for advancing protein engineering and design strategies.