A Virtual Mixture Approach to the Study of Multistate Equilibrium: Application to Constant pH Simulation in Explicit Water

Chemical and thermodynamic equilibrium of multiple states is a fundamental phenomenon in biology systems and has been the focus of many experimental and computational studies. This work presents a simulation method to directly study the equilibrium of multiple states. This method constructs a virtual mixture of multiple states (VMMS) to sample the conformational space of all chemical states simultaneously. The VMMS system consists of multiple subsystems, one for each state. The subsystem contains a solute and a solvent environment. The solute molecules in all subsystems share the same conformation but have their own solvent environments. Transition between states is implicated by the change of their molar fractions. Simulation of a VMMS system allows efficient calculation of relative free energies of all states, which in turn determine their equilibrium molar fractions. For systems with a large number of state transition sites, an implicit site approximation is introduced to minimize the cost of simulation. A direct application of the VMMS method is for constant pH simulation to study protonation equilibrium. Applying the VMMS method to a heptapeptide of 3 ionizable residues, we calculated the pKas of those residues both with all explicit states and with implicit sites and obtained consistent results. For mouse epidermal growth factor of 9 ionizable groups, our VMMS simulations with implicit sites produced pKas of all 9 ionizable groups and the results agree qualitatively with NMR measurement. This example demonstrates the VMMS method can be applied to systems of a large number of ionizable groups and the computational cost scales linearly with the number of ionizable groups. For one of the most challenging systems in constant pH calculation, SNase Δ+PHS/V66K, our VMMS simulation shows that it is the state-dependent water penetration that causes the large deviation in lysine66’s pKa.Author Summary: Computer simulation plays an important role to understand molecular systems and has been applied to problems of increasing complexity. Multistate equilibrium is a fundamental concept behind the structure and function of biological systems. Due to the limit in computing resources and lack of good alternative methods, computer simulation has been conducted for systems in a single state, sampling from one state to another to infer equilibrium properties. This sequential approach has been successful in many cases such as protonation equilibrium with implicit solvation model. However, state transition is difficult when explicit solvent is used for more accurate solvation description. Many efforts have been dedicated to overcome this difficulty. Analogous to real multistate systems, we proposed a virtual mixture of multiple states (VMMS) to directly simulate the equilibrium. State transitions are replaced by changes in state molar fractions. Mimicking a test tube environment, all states are simulated in parallel to equilibrate with each other. Application to constant pH simulation in explicit water demonstrates the capability of this method. It is expected that the VMMS method will find more applications in biological problems related to the equilibrium of competing states.