Regulatory mechanisms of phosphorylation in ovalbumin self-assembly for aggregates formation: Insights into size, structure, and morphology

In this study, the impact of different phosphorylation degrees on ovalbumin (OVA) assemblies was investigated by introducing negatively charged phosphorylated groups as driving forces at pH 9.0 and 85 degrees C for 2, 4, 6, 10, and 12 h to examine their turbidity, zeta-potential, and average particle size during phosphorylation-mediated aggregation. The presence of phosphate groups in the aggregates was confirmed by 31P NMR and FTIR spectra. The aggregation process, including amyloid formation, was analyzed by evaluating active thiol groups, intrinsic fluorescence spectra, surface hydrophobicity, circular dichroism, and thioflavin T (THT) fluorescence. TEM and small-angle scattering analysis of the morphological evolution of the self-assembly process over time revealed a structural progression from flexible to longer and thicker flexible fibrils, with the formation of shorter, more rigid semiflexible aggregates after 10h of phosphorylation. When phosphorylated for 12 h, the OVA aggregates exhibited a compact core primarily composed of cross beta-structures and surrounded by loosely packed protein segments with random coil structures. Overall, this study highlighted the enhancement of OVA selfassembly by phosphorylation, where electrostatic and hydrophobic interactions play pivotal roles in regulating the formation of protein self-assembly aggregates.