Dynamics of Ca(II)-Induced Aggregation of β-Casein as an Intrinsically Disordered Protein: Effect of Ca(II)...

The aggregation of intrinsically disordered proteins (IDPs) is of significant interest due to its role in proteopathies and nutritional and pharmaceutical potential. This study investigates the mechanism of Ca(II)-induced aggregation of an IDP β-casein (β-CN) using dynamic light scattering, cryo-transmission electron microscopy, and molecular dynamics simulations. Upon Ca(II) addition, β-CN undergoes successive induction, growth, and saturation phases, forming amorphous aggregates. Aggregation kinetics are highly dependent on the Ca(II) concentration. At a molar ratio exceeding 5:1, the hydrodynamic diameter of β-CN increased from 20 nm (oligomer) to >100 nm (aggregate product) in a minute. The induction phase is driven by neutralizing the phosphorylated groups via Ca(II) binding, while subsequent growth and saturation phases are governed by agglomeration of intermediate aggregates with nuclei-exposed oligomers, eventually forming aggregate products after conformational relaxation. We demonstrate that the porosity and tunable assembly of β-CN aggregates enable efficient encapsulation of bioactive molecules, offering promising applications in nanonutrition and nanotheranostics.