Hydronium Ions Accompanying Buried Acidic Residues Lead to High Apparent Dielectric Constants in the Interior of Proteins.

Internal ionizable groups are known to play important roles in protein functions. A mystery that has attracted decades of extensive experimental and theoretical studies is the apparent dielectric constants experienced by buried ionizable groups, which are much higher than values expected for protein interiors. Many interpretations have been proposed, such as water penetration, conformational relaxation, local unfolding, protein intrinsic backbone fluctuations, etc. However, these interpretations conflict with many experimental observations. The virtual mixture of multiple states (VMMS) simulation method developed in our lab provides a direct approach for studying the equilibrium of multiple chemical states and can monitor p Ka values along simulation trajectories. Through VMMS simulations of staphylococcal nuclease (SNase) variants with internal Asp or Glu residues, we discovered that cations were attracted to buried deprotonated acidic groups and the presence of the nearby cations were essential to reproduce experimentally measured p Ka values. This finding, combined with structural analysis and validation simulations, suggests that the proton released from a deprotonation process stays near the deprotonated group inside proteins, possibly in the form of a hydronium ion. The existence of a proton near a buried charge has many implications in our understanding of protein functions.