Constant-pH (CpHMD) simulations were performed using a recent GROMACS 2021 implementation with a modified CHARMM36m force field. This approach is based on the λ-dynamics method developed by Brooks and co-corkers . The system setup was similar to that discussed in the above section; however, to agree with the recommended CpHMD setup, both the leapfrog integrator and velocity rescaling with a 0.5 ps coupling time, as well as a PME Fourier spacing of 0.14 nm were used. Because only aspartate and glutamate were considered, a single-site representation (i.e., the proton can be bound to only one heavy atom in the residue) was employed; here, the A and B states represent the protonated and deprotonated forms of the titratable residue. The mass of the λ particle was set at 5 AU, and its temperature was maintained at 300 K using velocity rescaling with a 2 ps coupling time.