Ion Pairs Buried in Hydrophobic Environments within Proteins: Electrostatic Crosstalk between Buried Groups

Abstract

Internal ion pairs buried in the hydrophobic interior of proteins are essential for processes such as H+ transport, e- transfer, and catalysis. Despite their importance, the properties of buried ion pairs remain poorly understood. It has been suggested that medium- or long-range Coulomb interactions could play a role in tuning biological function, but this would only be possible if proteins behaved as materials with low dielectric constant, as assumed in many continuum electrostatics models. Buried ion pairs are of interest then not only for their functional roles, but also because they can be used to examine the balance between Coulomb and hydration energies in the dry, hydrophobic interior of proteins. As the distance between a pair of internal ionizable groups increases and the energy of their Coulomb interaction decreases, the balance of these energies will favor burial of the groups in the neutral state, eliminating any direct electrostatic interaction between them. To examine the magnitude of electrostatic crosstalk between buried ionizable groups experimentally, many variants were engineered on a highly stable form of staph nuclease. All variants have His66 in the hydrophobic core and either Asp or Glu residues buried at another internal position. Many His-Asp/Glu pairs were studied, and in no case were medium- or long-range Coulomb interactions observed. When the groups were within H-bond distance, strong, favorable coupling energies were measured, but only when the side chains of the buried pair could achieve a geometry favorable for an H-bond interaction. These data suggest that at short range the buried pair behaved as a strong hydrogen bond, but no evidence was found that they ever behave as titratable point charges interacting at a distance through Coulomb forces enhanced by the low dielectric protein medium.