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

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 Delta H degrees [-(14.19 +/- 2.56) kJ mol-1] and positive Delta S degrees [+(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 Delta H degrees [+(9.96 +/- 4.26) kJ mol-1] and Delta S degrees [+(120.05 +/- 14.14) J K-1mol-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 HEWLesculetin complexes, respectively, were also measured. Circular dichroism (CD) studies revealed that the % alpha-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 Tm value for the protein-ligand complexes (71.8 degrees C) compared to the native protein (73.3 degrees 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. (c) 2022 Elsevier B.V. All rights reserved.

Automated summary

In this study, multi-spectroscopic and computational methods were used to investigate the binding mechanism of esculin and esculetin with hen egg white lysozyme (HEWL). The results showed that both compounds interacted with HEWL through a static quenching mechanism with high binding constants. Hydrophobic forces and hydrogen bonding were involved in the binding process, leading to conformational changes and altered enzyme activity of HEWL.