∆+PHS Staphylococcal nuclease (SNase) variant (PDB: 3BDC ) and protein deglycase DJ-1 (PDB: 1P5F ), were taken from the PDB database. A double system in a single box setup was used, with a 3 nm distance between the protein and peptide (ACE-AXA-NH 2 ); this ensured charge neutrality during the alchemical transition. To prevent consequential protein-peptide interactions, a single Cα in each molecule was positionally restrained. We used the CHARMM36m 52 (with CHARMM-modified TIP3P 53 ) force field. A salt concentration consistent with the experimental setup was used. If no salt concentration was reported only K + or Cl -counterions were added. For all systems, an initial minimization using the steepest descent algorithm was performed. A constant temperature corresponding to the reference experimental setup was maintained implicitly using the leap-frog stochastic dynamics integrator with an inverse friction constant of γ = 0.5 ps -1 . The pressure was maintained at 1 bar using the Parrinello-Rahman barostat with a coupling time constant of 5 ps. The integration time step was set to 2 fs. Long-range electrostatic interactions were calculated using the Particle-mesh Ewald method with a real-space cut-off of 1.2 nm and grid spacing of 0.12 nm. Lennard-Jones interactions were force-switched off between 1.0 and 1.2 nm. Bonds to hydrogen atoms were constrained using the Parallel LINear Constraint Solver. To improve sampling, systems were run for 50 ns in 4 independent replicas, and the first 10 ns of each simulation was discarded as equilibration. From the remaining 40 ns, 400 non-equilibrium transitions of 500 ps each were generated and work values from the forward and backward transitions were collected using thermodynamic integration. These values were then used to estimate the corresponding free energy difference with Bennett's acceptance ratio as a maximum likelihood estimator relying on the Crooks fluctuation theorem. Bootstrapping was used to estimate the uncertainties of the free energy estimates, and these were propagated when calculating ∆∆G values.