where N prot and N deprot are the total number of frames in which the site is protonated and deprotonated, respectively. For titratable sites modeled in the single-site representation, we considered it protonated if λ is below 0.2, and deprotonated if λ is above 0.8. For sites that are described with the multisite description, we considered a state protonated if the λ associated with the protonated form of the residue is above 0.8, and deprotonated if the λ associated with the deprotonated form of the residue is above 0.8.