Free energy calculations for protein design

Free energy calculations have become invaluable in protein design, offering a powerful means to rapidly and accurately screen potential variants. Here, we provide a step-by-step protocol that combines molecular dynamics simulations with non-equilibrium alchemical free energy methods to tackle open questions in protein design. For system setup, simulation, and free energy analysis, our workflow relies on two free and open-source packages: pmx and GROMACS. Using the well-characterized protein switch MAD2, we demonstrate how to predict mutation-induced changes in protein behavior. At each step in the protocol, we highlight critical factors for obtaining high-quality free energy estimates and discuss common pitfalls and strategies to avoid them. Finally, we outline how this protocol can be extended to more challenging applications, such as charge-changing mutations, large-scale mutational scans, and allosteric coupling studies.