Sorbitol modulates the structure and nanomechanics of κ-casein amyloid fibrils.

Amyloid fibrils are highly ordered protein aggregates that, beyond their pathological roles, are increasingly recognized as functional structures in food systems. κ-casein is a particularly interesting model due to its dual relevance: it stabilizes casein micelles in milk but can also form amyloid fibrils under destabilizing conditions. In this work, we investigate how the food-grade osmolyte sorbitol modulates the fibrillation, structure, and nanomechanics of κ-casein. Thioflavin T fluorescence assays revealed that sorbitol accelerates fibril formation by eliminating the initial lag phase while maintaining similar elongation kinetics. Atomic force microscopy demonstrated that fibrils formed above 500 mM sorbitol become shorter and significantly stiffer, indicating tighter molecular packing. Complementary small-angle X-ray scattering analysis confirmed that these morphological changes occur without major alterations in the fibril cross-sectional architecture. All-atom molecular dynamics simulations showed that sorbitol induces a redistribution of conformational flexibility, promoting local compaction around central residues and enhanced mobility at the termini, leading to globally more extended conformations. Together, these results demonstrate that sorbitol acts as a molecular modulator of κ-casein aggregation, linking solution conditions to fibril morphology and nanomechanical properties. These findings provide molecular-level insight into how polyols influence protein self-assembly in food systems and highlight how osmolytes can be used to tune the structural and mechanical properties of amyloid fibrils formed by dietary proteins.