Disruption of metal ion homeostasis can lead to oxidative stress, which promotes protein misfolding and aggregation linked to several pathological conditions. The aim of our research was to investigate the influence of copper (Cu$^{2+}$) ions on the binding of N-acetyl-D-lactosamine (LacNAc) to hen egg white lysozyme (HEWL). UV-Vis and fluorescence spectroscopy were used to monitor the interaction between LacNAc, Cu$^{2+}$ ions and HEWL. The effect of copper ions on the secondary structure and the thermostability of HEWL were assessed using circular dichroism (CD) spectroscopy and differential scanning calorimetry (DSC), respectively. Molecular docking was used to predict the location of the binding site of LacNAc on HEWL. Fluorescence emission spectra analysis confirmed the binding of LacNAc to HEWL and indicated that Cu$^{2+}$ ions modulate the interaction between LacNAc and HEWL. Molecular docking predicted that LacNAc binds in the active site cleft of HEWL. The electrostatic potential map for HEWL showed an area with negative potential within the active site cleft, which could be a possible binding site for Cu$^{2+}$ ions. Cu$^{2+}$ ions do not cause substantial changes in the protein’s secondary structure under the conditions studied and they do not significantly destabilize HEWL. From these results, we suggest that Cu$^{2+}$ ions modulate the binding of LacNAc by competing for a common binding site.
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