The constant pH simulations of the aforementioned systems require reference states for Asp, Glu and His, in which the proton affinity (pK a ) is known from experiment. The measured and calculated (force field) deprotonation free energies of these reference states were used to include the effect of the pH bath, as well as the effects of the breaking and forming of chemical bonds in the simulation, i.e., V MM in Equation . The measured reference pK a values used in this work are included in Table . Note that the experimental values were obtained for pentapeptides, while tripeptides were used for computing V MM . This however did not affect the results, as shown in Figure of SI.