Kappa casein, a major protein component of milk, is capable of forming amyloid-like fibrils under defined conditions, a phenomenon with broad implications in both food science and human health. Yet, the molecular mechanism governing fibril formation remains incompletely understood, particularly within biologically relevant crowded environments. In this study, we explored the structural and mechanical features of kappa casein fibrils formed in the presence of sorbitol, serving as a mimic of molecular crowding. By integrating small-angle X-ray scattering (SAXS) with all-atom molecular dynamics (MD) simulations, we obtained a comprehensive view of fibril morphology and stability. SAXS analysis revealed a reduced persistence length, pointing to enhanced flexibility in sorbitol-induced fibrils compared to those formed under control conditions. Complementary AFM experiments highlighted changes in fibril stiffness, providing nanoscale insights into their mechanical behavior. In parallel, MD simulations allowed us to probe the atomistic details of fibril organization, while thermodynamic analyses derived from the simulations shed light on the energetic contributions of sorbitol to fibril stabilization and flexibility. Together, these approaches demonstrate that crowding agents can finely tune the balance between structural rigidity and conformational plasticity of amyloid fibrils. Looking ahead, we are investigating the biological implications of these altered fibril properties, focusing on their impact on cell viability and cytotoxicity in HeLa cells. Establishing the connection between fibril mechanics, thermodynamic stability, and cellular responses could provide fundamental insights into the role of protein aggregates in disease and guide the development of therapeutic or food-related strategies to modulate fibril formation.
Bence Fehér (2026) studied this question.