Effect of Water–DMSO Binary Solvent Mixture on the Behavior of an Intrinsically Disordered Protein, β-Casein

The study of protein behavior in water-dimethyl sulfoxide (DMSO) solvent mixture is of great interest in biophysical research, as DMSO is a widely used cosolvent in various experimental assays. However, investigating the behavior of intrinsically disordered proteins (IDPs) in a water-DMSO binary solvent mixture is still in its infancy. Herein, we present a comprehensive analysis of the behavior of β-casein (βCN), an IDP, in water-DMSO mixtures using a combination of steady-state fluorescence, time-resolved anisotropy, infrared spectroscopy, and fluorescence correlation spectroscopy (FCS), characterizing the protein stability and conformational dynamics. FCS analysis with Alexa-488-labeled βCN, revealed the variations in hydrodynamic radius with varying content of DMSO, signifying constrained mobility and expanded size of the protein. Further, fluorescence self-quenching of the fluorophore (Alexa-488) was analyzed to probe the conformational dynamics of βCN, with changes in the time constant suggesting protein unfolding followed by an assembly at higher DMSO content. These results were corroborated by infrared spectroscopy, where an increase in the amide-I absorption band indicated the formation of intermolecular antiparallel β-sheet aggregates. At higher DMSO concentrations, additional structural features were observed, which were further examined using optical and field emission scanning electron microscopy (FESEM).