Investigating the unique features of self-assembled hen egg white lysozyme nano-aggregates using biophysical approaches

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We report a novel strategy for synthesis of Hen-egg-white Lysozyme nanoparticles by alkaline pH induced aggregation at room temperature (298 K). Aggregation at alkaline pH is characterized by an absence of lag phase or nucleation phase. Thus, aggregation proceeds without any requirement of critical concentration of the nucleus. These nanostructures offer the advantage of synthesis in a size controlled manner. Intermolecular association by disulfide linkage (s-s) renders the protein conformation to be further stable against any electrostatic repulsion faced by the oligomeric units when transferred from pH 12.2 to pH 7.0. Detailed characterization of a complex self-assembling system presents an enormous challenge due to the heterogeneous nature of oligomeric species involved in the aggregation of HEWL. Biophysical methods like steady state fluorescence anisotropy, AFM, TEM, SAXS and DLS were employed to characterize the nanoparticles. In order to address the issue of size heterogeneity, chromatographic methods such as ion exchange and size exclusion have been used. Accessible hydrophobic regions in HEWL nanoparticles were assessed by ANS and Tryptophan fluorescence.
Supervisor: Rajaram Swaminathan