PREDICTION AND VALIDATION OF NARINGENIN – BOVINE LACTOFERRIN BINDING BY DOCKING, MOLECULAR DYNAMICS, AND ISOTHERMAL TITRATION NANOCALORIMETRY

Naringenin (NG) is a flavanone mainly found in grapefruits and citrus fruits. NG has potential for application in food products with bioactive appeal due to antioxidant, anti-inflammatory, and antidiabetic properties. Therefore, studying NG binding with bioactive food proteins is an important scientific and technologic issue. The aim of this work was to characterize thermodynamically the NG binding with bovine lactoferrin (bLF) using docking simulations (DS), molecular dynamics (MD), and isothermal titration nanocalorimetry (ITC). The prediction of the NG-bLF binding mode was performed by docking analysis using AutoDock Vina. The best model was also used in molecular dynamics carried out in NAMD program. To validate the predicted data, ITC experiments were carried out in a TAM III isothermal titration nanocalorimeter, where successive titrations of NG solution into a cell containing a bLF dispersion were made at pH 7.4. The best model of DS results predicted a standard Gibbs free energy change (ΔG°) of -32.22 kJ/mol in a protein pocket enriched of hydrophobic amino acids, and MD results suggested that only one NG molecule binds to each protein with the predicted affinity of -31.84 kJ/mol. On the other hand, the second naringenin moves away from the binding site after 50% of the simulation. These results were confirmed by ITC data: from a binding constant of 7.21 x 105 L/mol, the ΔG° value obtained was -33.42 kJ/mol. The complex stoichiometry obtained was 1.01, which also confirmed the predicted results that there was only one NG. The ITC results also demonstrated that the NG-bLF complex formation occurred with a major entropic contribution (ΔH° = -2.61 kJ/mol; TΔS° = 30.81 kJ/mol), which corroborates with the binding location suggested by MD. The results presented here can direct the NG application into food products using bLF as a natural vehicle.