Mutual interactions in a ternary protein/bioprotectant/water system

The dynamics of hydration water play a key role in many biological processes. The activity and function of proteins are strongly affected by the presence of water, which interacts primarily by means of hydrogen bonding. These interactions are examined in this work by a comparison of neutron vibrational spectra (Inelastic Neutron Scattering, INS) of dry lysozyme and hydrated lysozyme at h = 0.7 (g of H₂O/g of protein) with those of a lysozyme/water mixture at the same hydration value in the presence of the glass-forming bioprotectant trehalose. A difference spectrum, obtained by subtracting the dry lysozyme spectrum from that of the lysozyme/water mixture, yields the hydration water spectrum which is compared to the INS spectra of different kinds of ice in order to determine the changes induced by lysozyme on the hydrogen-bonded network of water. An additional comparison is performed by using a double-difference spectrum obtained by subtracting both the dry lysozyme and the trehalose spectra from the lysozyme/trehalose/water ternary spectrum. The effects of the mutual interactions among the three components, i.e. protein, disaccharide and water, are determined by comparison of the spectra of the dry systems (lysozyme, trehalose) with the difference spectra obtained from subtraction of the dry systems from the binary systems. It is concluded that the interfacial water more strongly affects the intermolecular mode region at low frequencies, whereas the vibrational spectra at high frequencies are more influenced by lysozyme and trehalose.