The role of water in the reversibility of thermal denaturation of lysozyme in solid and liquid states

Abstract

Although unfolding of protein in the liquid state is relatively well studied, its mechanisms in the solid state, are much less understood. We evaluated the reversibility of thermal unfolding of lysozyme with respect to the water content using a combination of thermodynamic and structural techniques such as differential scanning calorimetry, synchrotron small and wide-angle X-ray scattering (SWAXS) and Raman spectroscopy. Analysis of the endothermic thermal transition obtained by DSC scans showed three distinct unfolding behaviors at different water contents. Using SWAXS and Raman spectroscopy, we investigated reversibility of the unfolding for each hydration regime for various structural levels including overall molecular shape, secondary structure, hydrophobic and hydrogen bonding interactions. In the substantially dehydrated state below 37 wt% of water the unfolding is an irreversible process and can be described by a kinetic approach; above 60 wt% the process is reversible, and the thermodynamic equilibrium approach is applied. In the intermediate range of water contents between 37 wt% and 60 wt%, the system is phase separated and the thermal denaturation involves two processes: melting of protein crystals and unfolding of protein molecules. A phase diagram of thermal unfolding/denaturation in lysozyme - water system was constructed based on the experimental data.

Lysozyme–Sucrose Interactions in the Solid State: Glass Transition, Denaturation, and the Effect of Residual Water

“…These examples demonstrate that the model provides a reasonable fit to the experimental data; moreover, the obtained parameters such as ellipsoid radii and volume fractions are physically meaningful. For example, the ellipsoid radii at temperatures below denaturation are close to the ellipsoid parameters obtained in the liquid . This suggests that the protein molecules in the solid state in the presence of sucrose retain shapes close to their native shape (observed in aqueous solutions).…”

“…Figure shows the denaturation peak of lysozyme at different sucrose concentrations both for samples with residual moisture as well as dried samples. The T d in freeze-dried mixtures is higher than in lysozyme–water mixtures in the solid or liquid state . The calorimetric enthalpy (Table ) has similar values to the enthalpy in aqueous solution .…”

“…Figure ), which indicates an irreversible process, and the kinetic approach should be used to analyze experimental results . It was shown in our previous study that water activity ( a w ) has a big impact on lysozyme denaturation in the solid state. While solvation can stabilize the native state of protein molecules, it can also stabilize the unfolded molecules.…”

“…At a high saccharide-to-protein ratio, high-frequency bands prevail, which indicates a higher number of sugar–water interactions than in the previous case. The size and shape of protein molecules in the solid state with and without excipients have been extensively studied by X-ray and neutron scattering techniques. ,− In our previous work, we followed the structural changes in the lysozyme–water system using X-ray scattering experiments. We showed that the position of the protein–protein correlation peak, which appears between 2.09 and 2.61 nm –1 , shifts to lower values upon hydration and interpreted this behavior as the swelling of the material.…”

Creation of Cross-Linked Crystals With Intermolecular Disulfide Bonds Connecting Symmetry-Related Molecules Allows Retention of Tertiary Structure in Different Solvent Conditions

“…From these crystals, no X-ray diffractions were observed under cryogenic conditions, indicating their loss of diffraction ability throughout the soaking operation. Because the crystal shapes were maintained during a week of soaking, the acid/base denaturation of proteins in the cross-linked crystals, resulting in the amorphous solid state, could be limited to their partial unfolding ( Phan-Xuan et al, 2021 ). Indeed, pH is known to influence the stability of proteins by altering the net charge on the proteins, and many proteins denature at extremes of pH because of the presence of destabilizing intramolecular electrostatic interactions ( Fink et al, 1994 ; Khurana et al, 1995 ; Dwyer et al, 2000 ).…”