Protein Salting Out Observed at an Air-Water Interface

A protein salting-out process is directly observed at an air-water interface. By using time-resolved X-ray specular reflectivity and off-specular diffuse scattering, we identified several key stages in the adsorption of hen egg white lysozyme in a concentrated NaCl solution, (1) adsorption-induced unfolding, (2) monolayer formation with unfolded proteins, (3) protein refolding, and (4) island formation with the refolded proteins. Stages 3 and 4 are not observed either at the isoelectric point or in the salt-free solution, suggesting that they are induced by screening of the positive charges in the lysozyme by chloride ions. It is considered that the hydrated salt ions act to minimize the water-accessible surface area of the protein, not only enhancing protein dehydration (stages 1 and 2) but also assisting in protein refolding and association (stages 3 and 4). These results provide insight into the early stages of protein crystal nucleation.