Determinationof Ion Atmosphere Effects on the NucleicAcid Electrostatic Potential and Ligand Association Using AH+·C Wobble Formation in Double-Stranded DNA

摘要

The high charge density of nucleic acids and resulting ion atmosphere profoundly influence the conformational landscape of RNA and DNA and their association with small molecules and proteins. Electrostatic theories have been applied to quantitatively model the electrostatic potential surrounding nucleic acids and the effects of the surrounding ion atmosphere, but experimental measures of the potential and tests of these models have often been complicated by conformational changes and multisite binding equilibria, among other factors. We sought a simple system to further test the basic predictions from electrostatics theory and to measure the energetic consequences of the nucleic acid electrostatic field. We turned to a DNA system developed by Bevilacqua and co-workers that involves a proton as a ligand whose binding is accompanied by formation of an internal AH⁺·C wobble pair [Siegfried, N. A., et al. Biochemistry, >2010, 49, 3225]. Consistent with predictions from polyelectrolyte models, we observed logarithmic dependences of proton affinity versus salt concentration of −0.96 ± 0.03and −0.52 ± 0.01 with monovalent and divalent cations,respectively, and these results help clarify prior results that appearedto conflict with these fundamental models. Strikingly, quantitationof the ion atmosphere content indicates that divalent cations arepreferentially lost over monovalent cations upon A·C protonation,providing experimental indication of the preferential localizationof more highly charged cations to the inner shell of the ion atmosphere.The internal AH⁺·C wobble system further allowed usto parse energetic contributions and extract estimates for the electrostaticpotential at the position of protonation. The results give a potentialnear the DNA surface at 20 mM Mg²⁺ that is much less substantialthan at 20 mM K⁺ (−120 mV vs −210 mV). Thesevalues and difference are similar to predictions from theory, andthe potential is substantially reduced at higher salt, also as predicted;however, even at 1 M K⁺ the potential remains substantial,counter to common assumptions. The A·C protonation module allowsextraction of new properties of the ion atmosphere and provides anelectrostatic meter that will allow local electrostatic potentialand energetics to be measured within nucleic acids and their complexeswith proteins.