Origin of pKa Shifts of Internal Lysine Residues in SNase Studied Via Equal-Molar VMMS Simulations in Explicit Water.

Protein internal ionizable groups can exhibit large shifts in pKa values. Although the environment and interaction changes have been extensively studied both experimentally and computationally, direct calculation of pKa values of these internal ionizable groups in explicit water is challenging due to energy barriers in solvent interaction and in conformational transition. The virtual mixture of multiple states (VMMS) method is a new approach designed to study chemical state equilibrium. This method constructs a virtual mixture of multiple chemical states in order to sample the conformational space of all states simultaneously and to avoid crossing energy barriers related to state transition. By applying VMMS to 25 variants of staphylococcal nuclease with lysine residues at internal positions, we obtained the pKa values of these lysine residues and investigated the physics underlining the pKa shifts. Our calculation results agree reasonably well with experimental measurements, validating the VMMS method for pKa calculation and providing molecular details of the protonation equilibrium for protein internal ionizable groups. Based on our analyses of protein conformation relaxation, lysine side chain flexibility, water penetration, and the microenvironment, we conclude that the hydrophobicity of the microenvironment around the lysine side chain (which affects water penetration differently for different protonation states) plays an important role in the pKa shifts.