GROMACS 2023 was used to run all simulations. For all systems, an initial minimization was performed using the steepest descent algorithm. A constant temperature corresponding to the reference experimental setup was maintained implicitly using the leap-frog stochastic dynamics integrator 36,37 with a 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. Production simulations were 50 ns in length and run in quadruplicate. The first 10 ns of simulation was discarded as equilibration and from the remaining 40 ns, 400 non-equilibrium transitions of 200 ps were generated. Work values from the forward and backward transitions were collected using thermodynamic integration and these were used to estimate the corresponding free energy 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.