Binding and thermodynamics of R EV peptide−ct DNA interaction

Abstract The thermodynamics of DNA‐ligand binding is important as it provides useful information to understand the details of binding processes. HIV‐1 REV response element (RRE) located in the env coding region of the viral genome is reported to be well conserved across different HIV‐1 isolates. In this study, the binding characteristics of Calf thymus DNA (ctDNA) and REV peptide from HIV‐1 were investigated using spectroscopic (UV–visible, fluorescence, and circular dichroism (CD)) and isothermal titration calorimetric (ITC) techniques. Thermal stability and ligand binding properties of the ctDNA revealed that native ctDNA had a T m of 75.5 °C, whereas the ctDNA–REV peptide complex exhibited an incremental shift in the T m by 8 °C, indicating thermal stability of the complex. CD data indicated increased ellipticity due to large conformational changes in ctDNA molecule upon binding with REV peptide and two binding stoichiometric modes are apparent. The ctDNA experienced condensation due to large conformational changes in the presence of REV peptide and positive B→Ψ transition was observed at higher molar charge ratios. Fluorescence studies performed at several ligand concentrations revealed a gradual decrease in the fluorescence intensity of EtBr‐bound ctDNA in response to increasing ligand concentrations. The fluorescence data further confirmed two stoichiometric modes of binding for ctDNA–REV peptide complex as previously observed with CD studies. The binding enthalpies were determined using ITC in the temperature range of 293 K–308 K. The ITC binding isotherm was exothermic at all temperatures examined, with low Δ H values indicating that the ctDNA–REV peptide interaction is driven largely by entropy. The heat capacity change (Δ C p ) was insignificant, an unusual finding in the area of DNA‐peptide interaction studies. The variation in the values obtained for Δ H , Δ S , and Δ G with temperature further suggests that ctDNA–REV peptide interaction is entropically driven. ITC based analysis of salt dependence of binding constant gave a charge value ( Z ) = +4.01, as determined for the δln K /δln[Na + ] parameter, suggesting the participation of only 3–4 Arg out of 11 Arg charge from REV peptide. The stoichiometry observed for the complex was three molar charge of REV peptide binding per molar charge of ctDNA. ITC based analysis further confirmed that the binding between ctDNA and REV peptide is governed by electrostatic interaction. Molecular interactions including H‐bonding, van der Waals forces, and solvent molecules rearrangement, underlie the binding of REV peptide to ctDNA.