To improve sampling, systems were run for 25 ns in 4 independent replicas; in each case the first 5 ns was discarded as equilibration. From the remaining 20 ns, 200 non-equilibrium transitions of 200 ps 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 with Bennett's acceptance ratio 119 as a maximum likelihood estimator relying on the Crooks Fluctuation Theorem 120 . Bootstrapping was used to estimate the uncertainties of the free energy estimates , and these were propagated when calculating the ∆∆G values. Equation 1 was used to convert between ∆∆G and pK a (protein) values using the corresponding references (i.e., aspartate: 3.94±0.03, glutamate: 4.25±0.05, and lysine: 10.4±0.08) .