A change in the protonation state affects the total charge of the simulated system, which can lead to artifacts when Ewald summation is used to treat the electrostatic interactions. In our implementation of constant pH MD we avoid this problem by introducing titratable buffers into the simulation box that compensate for the charge fluctuations of the titratable residues. In the original implementation of constant pH MD in GROMACS, 10 the buffers were hydronium molecules that compensated the overall charge fluctuations by changing their charge between 0 and +1 e. To prevent sampling charges beyond this interval, a biasing potential with steep edges at λ = 0 and λ = 1 was introduced to restrict the sampling of λ.