Unfolding and aggregation of lysozyme: A thermodynamic and kinetic study by FTIR spectroscopy

The unfolding of hen egg-white lysozyme dissolved both in D2O and CH3CH2OD/D2O was studied by Fourier Transform Infrared (FTIR) absorption spectroscopy at different protein concentrations. A detailed description of the local and global rearrangement of the secondary structure upon a temperature increase, in the range 295 to 365 K, was obtained through the analysis of the amide I band. Thermodynamic parameters for the melting, and the effect of the co-solvent in determining a change in thermal stability of the protein were evaluated. The protein-protein interactions were also followed as a function of temperature: a strong dependence of the cluster stability and aggregation yield on the solvent composition was observed. Finally, FTIR spectra taken at successive time steps of the aggregation enabled intermolecular contacts to be monitored as a function of time, and kinetic information to be obtained showing that both unfolded and folded states of lysozyme act as reactants for the clustering event.

Research Highlights► We followed the effects of ethanol addition to lysozyme/water solutions. ► A reduced stability of the folded state was revealed on increasing ethanol fraction. ► An enhancement of intermolecular interactions between protein chains was observed upon increase of alcohol concentration. ► We evidenced an irreversible association initiated by the unfolded and the folded states of the macromolecule. ► We obtained different hydrogels under different thermal treatment of a single protein solution.