Hydration Dynamics in the Central Region of ClC-ec1

To calculate the time scale for the transition from a low to high hydration state in the central region of ClC-ec1, the low hydration state first needed to be created in the MS-RMD structure. To do this, a high hydration configuration was taken from the last MS-RMD snapshot at umbrella window ζ_R = 0.24 (Figure ​Figure66D). An additional harmonic potential was applied to the water density CV (N_w) in the predefined box (see refs (23) and (39) and for definition). The force constant of the harmonic potential was set to be 10 kcal·mol^–1, and the center of the potential was at N_w = 0. The center of the box was chosen to be the midpoint between the center of mass of each carboxyl group of E148 and E203, and the box size was set to be 2 × 2 × 2 ų, which covered the position of the second water. The second water present in the initial configuration was expelled from the central region by the harmonic potential centered at N_w = 0. The MS-RMD simulation was run for 200 ps, until the system was equilibrated in the absence of the second water.

Ten independent trajectories were initiated from the last MD frame of the simulation above, after the velocities of all atoms were randomized and the harmonic potential centered at N_w = 0 was released. The coordination number of the pivot hydronium was defined as the number of oxygen atoms (from water or the E203 carboxyl group) within 3 Å from the pivot hydronium oxygen. The time for the second water entering the central region was estimated by averaging the 10 simulations time to when there was a productive (not transient) transition of the coordination number from 2 to 3.