Thermal and pre-sheared induced aggregation of hen egg white lysozyme and the effect of Centella asiatica water extract on the aggregation

Protein aggregation is a pivotal phenomenon with profound implications in both biopharmaceutical development and physiological processes. Understanding its mechanisms and discovering effective strategies for its prevention are therefore of paramount importance. Hen Egg White Lysozyme (HEWL) was considered a model protein in this study, and the aggregation kinetics were assessed under thermal (65 °C and pH 2) and thermomechanical conditions. The effect of Centella asiatica water extract (CWE), a nootropic plant extract, was evaluated on the thermal-induced aggregation of HEWL. Pre-sheared HEWL samples, generated under combined thermal-mechanical stress and used as shear-induced seeds (SIS), exhibited a faster apparent aggregation rate of 0.97 day^-1, while non-seeded aggregates showed a rate of 0.87 day^-1. In the presence of CWE (100 μg/mL), no measurable sigmoidal aggregation was observed, indicating near-complete suppression of the aggregation process. This attenuation was confirmed through circular dichroism spectroscopy, and aggregate morphology was analyzed via Atomic Force Microscopy. CWE also demonstrated neuroprotective effects against HEWL aggregate-induced toxicity in IMR-32 neuroblastoma cells. Secondary structure analysis revealed a structural transition from an α-helix to an amyloid-like, β-sheet-rich conformation with a high degree of disorder, consistent with a type II β-protein. Conversely, the addition of CWE delayed this loss of α-helical content in a concentration-dependent manner. Stern-Volmer fitting indicated a static quenching mechanism, suggesting protein-ligand complex formation in the ground state. Molecular docking identified 1,4-Di-O-caffeoylquinic acid as the compound with the highest binding affinity (-8.6 kcal/mol). Molecular dynamics simulations highlighted the roles of van der Waals, electrostatic, and hydrophobic interactions in stabilizing the complex. The binding pocket, comprising E³⁵, D⁵², W⁶², W⁶³, and W¹⁰⁸, facilitated hydrogen bonding and included key residues of the HEWL active site. CWE also retained the native biofunctionality of HEWL, suggesting its potential as a stabilizing excipient against protein aggregation. Further studies using disease-relevant amyloidogenic proteins and therapeutic protein formulations are warranted to evaluate the broader translational applicability of CWE.