Abstract
Proteins in living organisms exist in complex aqueous solutions or embedded in membranes. In solution, proteins are surrounded by a tightly bound hydration layer, which is more ordered and less mobile than bulk water. As a consequence, water plays a major role in controlling protein structure stability, conformational flexibility, dynamics, and functionality, but it also appears that protein surface regulates the structuring of the surrounding water. The presence of cosolvents can modify the hydration layer characteristics and then the whole protein structural and dynamical properties. Because cytoplasm or biological liquids are complex solutions, the knowledge of the solvation shell characteristics in mixed solvents should be considered as a crucial step in describing biological processes at molecular level. This review reports on recent studies on the structural and thermodynamic properties of model proteins dissolved in binary solvent mixtures by small-angle neutron scattering (SANS) and differential scanning microcalorimetry (DSC) techniques. We will show that contrast variation SANS experiments allow to acquire a direct knowledge of both protein structure and protein solvation shell (in terms of low-resolution shape and solvent/cosolvent composition), while DSC experiments provide information on all the relevant thermodynamic properties. We will focus on two main points. First, an extended description of the thermodynamic model used to define the equilibria between water and cosolvent molecules in the protein solvation shell will be presented. Second, the determination of the peculiar characteristics of the protein solvation layer, which will be illustrated by considering different systems. As a conclusion, we will show that the investigation of structure and thermodynamics of proteins in binary aqueous mixtures is an important way to understand the role of hydration in protein stability and activity.