Binding of bioactive esculin and esculetin with hen egg white lysozyme: Spectroscopic and computational methods to comprehensively elucidate the binding affinities, interacting forces, and conformational alterations at molecular level

Abstract

The binding of bioactive esculin and esculetin with lysozyme have been studied using multi-spectroscopic and computational methods. Intrinsic fluorescence of lysozyme was quenched via static quenching mechanism by these ligands and binding was observed to be moderate in nature. Structural alteration of the protein was noted upon ligand binding and enzymatic activity of lysozyme was also influenced in vitro. • Coumarin derivatives, esculin and esculetin quenched intrinsic fluorescence of HEWL through a static quenching mechanism. • Trp residue occupied a more hydrophobic environment upon interactions with esculin and esculetin. • The α-helical content of HEWL decreased in presence of ligands which could have affected the enzymatic activity of HEWL. • Δ G ° values were observed to be negative which indicated complex formation occurred spontaneously. Multi-spectroscopic and computational methods were employed to investigate the binding mechanism of esculin and esculetin with hen egg white lysozyme (HEWL). UV–vis and fluorescence spectroscopy proved static quenching mechanism was involved in the fluorescence quenching process. The binding constant ( K b ) determined for HEWL-esculin/esculetin complex was in the order of 10 4 M −1 . A negative Δ H ° [-(14.19 ± 2.56) kJ mol −1 ] and positive Δ S ° [+(40.98 ± 3.10) J K −1 mol −1 ] for HEWL-esculin complex suggested the presence of hydrophobic forces and hydrogen bonding. Whereas positive values of both Δ H° [+(9.96 ± 4.26) kJ mol −1 ] and Δ S ° [+(120.05 ± 14.14) J K -1 mol −1 ] for HEWL-esculetin complex indicated the presence of hydrophobic forces in the binding process. Blue shifts were observed for HEWL-esculin/esculetin complexes on carrying out synchronous and three-dimensional (3-D) fluorescence. A red edge excitation shift (REES) of 3 nm was observed for HEWL, whereas 6 and 5 nm for HEWL-esculin and HEWL-esculetin complexes, respectively, were also measured. Circular dichroism (CD) studies revealed that the % α-helical content of HEWL (25.56 ± 2.24) decreased in the presence of esculin (17.96 ± 1.54) and esculetin (19.90 ± 2.14). Amide I peak of HEWL, determined from Fourier transform infrared (FTIR) studies, shifted from 1657 cm −1 to 1654 cm −1 and 1652 cm −1 upon complexation with esculin/esculetin, respectively. The binding distance ( r ) for HEWL-esculin (4.40 ± 0.14 nm) and HEWL-esculetin (4.66 ± 0.11 nm) complexes were determined from fluorescence resonance energy transfer (FRET) theory. Thermal analysis resulted in a lower T m value for the protein–ligand complexes (71.8 °C) compared to the native protein (73.3 °C). Molecular docking and molecular dynamic (MD) simulations provided insight into the binding affinity and flexibility of HEWL upon complexation, respectively. In the presence of esculin and esculetin, a change in enzyme activity of HEWL was observed.