.pdb at pH=4 and from an existing system at pH=4, barnase-barstar at pH=7 and GLIC at pH=4. For the parameterization of arginine, 1 ns simulations with λ-coordinates fixed at values ranging from -0.1 to 1.1 with 0.1 steps were first run to obtain an initial guess for the V MM . Then 10 replicas of 100 ns simulations with pH equal to pK a and biasing barrier set to 0 were run. From those sampling simulations, new coefficients for the V MM polynomial fit were calculated and another 10 replicas of 100 ns simulations with pH equal pK a and biasing barrier set to 0 with updated V MM were run.