Translational Diffusion and Self-Association of an Intrinsically Disordered Protein κ-Casein Using NMR with Ultra-High Pulsed-Field Gradient and Time-Resolved FRET.

Much attention has been given to studying the translational diffusion of globular proteins, whereas the translational diffusion of intrinsically disordered proteins (IDPs) is less studied. In this study, we investigate the translational diffusion and how it is affected by the self-association of an IDP, κ-casein, using pulsed-field gradient nuclear magnetic resonance and time-resolved Förster resonance energy transfer. Using the analysis of the shape of diffusion attenuation and the concentration dependence of κ-casein diffusion coefficients and intermolecular interactions, we demonstrate that κ-casein exhibits continuous self-association. When the volume fraction of κ-casein is below 0.08, we observe that κ-casein self-association results in a macroscopic phase separation upon storage at 4 °C. At κ-casein volume fractions above 0.08, self-association leads to the formation of labile gel-like networks without subsequent macroscopic phase separation. Unlike α-casein, which shows a strong concentration dependence and extensive gel-like network formation, only one-third of κ-casein molecules participate in the gel network at a time, resulting in a more dynamic and less extensive structure. These findings highlight the unique association properties of κ-casein, contributing to a better understanding of its behavior under various conditions and its potential role in casein micelle formation.