Studies on the Effect of Surface Functionalized Carbon Quantum Dots Towards Hen Egg White Lysozyme Amyloidogenesis

Abstract

Amyloids are stable, ordered, fibrillar aggregates formed by various polypeptide chains when subjected to specific environmental conditions. In vivo amyloid formation is induced by misfolding of the protein, which in turn, is influenced by mutations in the amino acid sequence. Additionally, the altered cellular environmental conditions, including a rise in temperature, high and/or low pH, oxidative stress, and increased glucose levels, make the protein lose its native conformation which eventually tend to partially unfold and aggregate. In vitro, amyloid formation can be triggered by altering certain external parameters such as pH, temperature, agitation etc. The phenomenon of protein aggregation is associated with pathological features and implicated in several neurodegenerative diseases including Alzheimer’s disease, Huntington’s disease, Amyotrophic lateral sclerosis, Parkinson’s disease, Type-II diabetes, prion diseases, and many others. Despite extensive research, an effective therapeutic strategy against amyloidosis remains elusive. In recent times, carbon quantum dots (CQDs) are attaining more importance and being implemented to modulate amyloid formation. CQDs possess an advantage over other inhibitors depending on properties, including ease of synthesis, size, optical properties, high surface area to volume ratio, ease of modifications, biocompatibility, and high quantum yield. The aim of the thesis includes, firstly, synthesis of CQDs from natural sources like kitchen spices mix powder, surface functionalization with negatively and positively charged carboxyl and amine functional groups respectively through hydrothermal treatment methods, and characterization of the as-synthesized CQDs. In the second part, efforts were made to elucidate effects of the bare and surface functionalized CQDs towards the model protein “hen egg white lysozyme” (HEWL), which is reported to form amyloid under in vitro conditions. HEWL is homologous to the human lysozyme which is responsible for hereditary systemic amyloidosis. The effects of the CQDs towards HEWL amyloid formation has been monitored using several biophysical, biochemical, and microscopic methods such as thioflavin T assay, transmission electron microscopy, dynamic light scattering assay, confocal microscopy, MTT assay and ROS assay. Finally, the underlying mechanism of action of the CQDs on HEWL was elucidated by monitoring their effects on the structure and stability of HEWL. Further, the mode of interactions between the CQDs and HEWL were analyzed using various spectroscopic approaches.