Buffer particles are used in constant pH MD to maintain the neutrality of the simulated system. Ideally, buffers should not introduce any artifacts due to binding to titratable groups, binding to each other, or penetrating into hydrophobic regions. To prevent such behavior, we optimized the charge range and Lennard-Jones parameters of the buffers. To this end, we performed a series of enhanced sampling simulations with the accelerated weight histogram method (AWH). In one set of simulations with two buffers in the simulation box (BUF 2 , Table ), we quantified the sampling efficiency from the friction metric, as a function of the absolute charge per buffer particle. In these simulations, the charge of one buffer was changed from 0 to +0.8, while simultaneously the charge of the other buffer was changed from 0 to -0.8 in order to maintain neutrality. The CHARMM36m