Experimental Evidence of Solvent-Separated Ion Pairs as Metastable States in Electrostatic Interactions of Biological Macromolecules

Electrostatic interactions via ion pairs are vital for biological macromolecules. Regarding the free energy of each ion pair as a function of the interionic distance, continuum electrostatic models predict a single energy minimum corresponding to the contact ion-pair (CIP) state, whereas atomically detailed theoretical hydration studies predict multiple energy minima corresponding to the CIP and solvent-separated ion-pair (SIP) states. Through a statistical analysis of high-resolution crystal structures, we present experimental evidence of the SIP as a metastable state. The histogram of interionic distances between protein side-chain NH 3 + and DNA phosphate groups clearly shows two major peaks corresponding to the CIP and SIP states. The statistical data are consistent with the probability distribution of the CIP–SIP equilibria previously obtained with molecular dynamics simulations. Spatial distributions of NH 3 + ions and water molecules around phosphates reveal preferential sites for CIP and SIP formations and show how the ions compete with water molecules.