Regulating aggregation of an intrinsically disordered chaperone-like casein.

Protein aggregation involving the conversion of soluble protein monomers into insoluble aggregates is prevalent in human diseases, food processing, food formulations, biotechnology-based therapeutics, etc. Molecular chaperones are typically globular proteins that regulate protein folding and aggregation. However, a unique chaperone-like milk protein namely, β-casein, is intrinsically disordered and prone to aggregation under physiological conditions. To regulate protein aggregation, there is a pressing need to devise strategic interventions that require a detailed understanding of the protein conformational changes during self-association. Here, we show that sodium chloride (NaCl) can modulate calcium ions (Ca)-induced spontaneous aggregation of β-casein under physiological conditions. Using fluorescence and Raman spectroscopy coupled with light scattering and transmission electron microscopy, we delineate the structural attributes of β-casein during Ca-mediated self-association. Our findings reveal that the binding of divalent Ca to five phosphorylated serine residues (calcium phosphate binding-short linear sequence motif; CaP-SLiM), located within the N-terminal-domain of β-casein, is an obligatory prerequisite. This binding event subsequently triggers the formation of inter-casein bridges that facilitate multivalent interactions between the hydrophilic, disordered β-caseins, driving the self-assembly wherein hydrophobic interactions are insignificant compared to β-casein-CaCl interactions. Further, the Ca-induced β-casein aggregation is accompanied by a disorder-to-order transition resulting in non-amyloid, spherical aggregates. We also demonstrate that NaCl influences the aggregation propensity of β-casein by electrostatically screening the polypeptide and leads to the formation of aggregation-incompetent oligomers by abolishing the binding of Ca to β-casein and the subsequent formation of inter-casein linkages, thus, affirming the pivotal role of CaP-SLiMs and multivalency during β-casein aggregation.