In the constant pH MD simulations, the mass of λ-particles was set to 5 atomic units, and temperature was kept constant at 300 K with a separate v-rescale thermostat for the λ-degrees of freedom, with a time constant of 2.0 ps -1 . The single-site representation, defined and described in the accompanying paper, 15 was used for Asp, Glu, Lys, C-ter, and N-ter, whereas the multisite representation, also described in that paper, was used for His. The same pH and biasing potentials were used as in Aho et al. In the sampling simulations of single titratable residues, the pH was set equal to the pK a , and the barrier height of the biasing potential was set to zero. The titration of the cardiotoxin V (PDB ID: 1CVO ) protein was performed by running ten independent replicas of 100 ns each, for 15 equidistantly spaced pH values in the range from 1.0 to 8.0, using both the original and modified CHARMM36m force field. In the titration simulations, the barrier height of the biasing potential was set to 7.5 kJ mol -1 for groups modeled with single-site representation, and to 5 kJ mol -1 for groups modeled with the multisite representation.