pmx 33 was used for system setup, hybrid topology generation, and analysis. Initial protein structures were taken from the PDB database and mutations were introduced using pdbfixer. In total we consider 12 proteins and 40 cysteine residues and 22 histidines (Table ). A double system in a single box setup was used, with a 3 nm distance between the protein and peptide (ACE-Ala-X-Ala-NH 2 ); this ensured a neutral box at every step of the alchemical transformation. To ensure that the protein and peptide did not interact, a single Cα in each molecule was positionally restrained. We used the CHARMM36m (with mTIP3P ) and Amber14SB (with TIP3P 32 ) force fields, both having previously performed well for simulations involving non-equilibrium alchemical calculations.