Vibrational Circular Dichroism Spectra of Lysozyme Solutions: Solvent Effects on Thermal Denaturation Processes

Vibrational spectroscopy has been applied to the study of conformational variation of lysozyme during thermal denaturation. An infrared and vibrational circular dichroism (VCD) analysis of lysozyme in D2O, D2O/EtOD, and D2O/DMSO at different solvent compositions and pH's was accomplished. Complete deuteration effects on amidic groups were revealed through the analysis of the amide I band of lysozytne dissolved in deuterated water. The comparison of IR absorption and VCD spectra of lysozyme in D2O revealed the higher sensitivity of the VCD technique to the presence of partially or fully deuterated secondary structure of lysozyme. Aggregation was induced by thermalization of lysozyme at different solvent compositions and pH values. Interestingly, IR and VCD showed different sensitivities to reveal intermolecular beta-sheet aggregation under thermal denaturation. The VCD intensity enhancement was observed as the aggregate dimension increased, whereas IR was sensitive to the nucleation step of the process. Moreover, chirality of supramolecular species was revealed through the analysis of the VCD sign pattern. For the first time, we demonstrate how aggregates produced under different solvent compositions but VCD sign pattern and consequently the same sense of growth. similar pH's showed the same VCD sign pattern and consequently the same sense of growth.