Complexation of Hen Egg White Lysozyme With Bioactive Phenolic Acids: Molecular Insights Into the Binding Mechanisms and Inhibitory Effects Against Protein Aggregation

Protein misfolding and aggregation are central to many protein aggregation disorders, highlighting the need for effective aggregation inhibitors. This study investigated fructose‐induced aggregation of hen egg‐white lysozyme (HEWL) and its modulation by the phenolic acids ferulic acid (FA), caffeic acid (CA) and chlorogenic acid (CGA) at 100 and 200 μM. Circular dichroism and ThT fluorescence assays showed that fructose promoted aggregation through increased β‐sheet content and fluorescence intensity, respectively, whereas all phenolic acids suppressed β‐sheet formation. CA displayed the highest aggregation inhibition activity, followed by FA and CGA. FESEM imaging confirmed fructose‐induced HEWL aggregation without fibril formation and demonstrated disruption of aggregates in the presence of phenolic acids. These findings were explained by other biophysical characterisation studies. Fluorescence spectroscopy indicated a static quenching mechanism for all complexes, with FA and CA displaying atypical quenching behaviour. Binding constants suggested moderate affinity, with CA (4.11 ± 0.57 × 10 4 M −1 at 300 K) showing the strongest interaction. +Δ S and +Δ H indicated hydrophobic interactions as the main stabilising force for all complexes, while computational studies revealed additional H‐bonding interactions within aggregation‐prone regions (K‐peptide) of HEWL. Together, experimental and computational findings demonstrate that these phenolic acids effectively modulate HEWL aggregation by stabilising native‐like conformations and disrupting aggregate formation.