Ionizable residues in enzyme active sites and the interior of ion transporters provide crucial stabilization of charged species in relatively nonpolar environments. Therefore, understanding their properties and physical factors that stabilize their presence in the protein interior is of great fundamental and practical importance, which is generally challenging based on experimental studies alone. Since these buried charges and/or dipoles are not well-solvated, it also remains unclear how explicit electronic polarization impacts the description of protein/ion-pair interactions.<br/><br/>Recent structural and thermodynamic studies of a series of variants of Staphylococcal nuclease (SNase) involving buried Glu-Lys pairs suggested that both the stability and the ionization state of the buried Glu-Lys pair are sensitive to its orientation. We have conducted extensive molecular dynamics simulations and free energy computations of several SNase mutants with different ionization states using both non-polarizable and polarizable force fields to further clarify the situation and provide insights on the molecular level. Free energy simulations with the CHARMM36 force field recapitulate the relative stability of Glu23-Lys36 (EK) vs Lys23-Glu36 (KE) mutants measured experimentally and support the reverse protonation of the buried Glu and Lys. Even though the general structural responses and hydration levels in the cavity are relatively consistent between the simulations with CHARMM36 or CHARMM Drude force field, explicit inclusion of electronic polarization and anisotropic charge distributions using the Drude oscillator model and off-center charges underscores the impact of reverse polarity of the buried Glu-Lys pairs due to different geometries and charge distributions on the stabilization of the ion pairs by cavity water and nearby protein residues. The current study demonstrates the essential roles of free energy simulations and explicit polarization in understanding the stabilization of buried titratable residues and the relevant energetic contributions in enzymes and bioenergetic systems. Our results and computational protocols thus form the basis for the rational design of functional buried charge networks in proteins and calibration of different polarizable models.

Stabilization of buried charges and ion pairs in proteins: Hydration and polarization

ACS Spring 2022

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