Modulation of Different Proteins’ Conformational Dynamics in The Presence of ZnO Nanoparticles with Varying Surface Properties

Abstract

In the recent years, with the advent of nanotechnology, nanoparticles have received immense attention in various fields including the medical and pharmaceutical industries. With increased applications, increases the risk of these nanoparticles’ exposure to the biological milieu. Hence it becomes imperative to understand the nanoparticle interaction at bio-interface especially with the protein molecules, as they are major soluble constituent of cytosol and tend to get adsorbed on nanoparticles as it enters the biological milieus. This interaction not only affects the nanoparticle properties but also induces changes in the protein conformation. Proteins are one of the most abundant and important biological molecules. A correctly folded conformation is required to execute its biological functions. However, due to various intrinsic (polypeptide sequence, mutation, presence of aggregation prone regions (APRs)) and extrinsic factors (environmental conditions) certain proteins have higher tendency to misfold into amorphous or fibrillar aggregates. This misfolding or aggregation of a protein leads to loss of its function; exert cellular toxicity and consequently onsets human disorders (e.g. Alzheimer’s disease, Huntington’s disease, Parkinson’s disease, amyotrophic lateral sclerosis and type II diabetes). Over the years, significant research interests have developed therapeutic application of nanoparticles in the protein aggregation and the diseases associated with amyloid. Due to their unique properties, nanoparticles (NPs) have shown significant effect on the protein conformation. However, the nanoparticle interaction with protein can act as a double edge sword depending on the strength of interactions, which in turn depend upon the physiochemical properties of both NPs and proteins interacting interfaces. The advantage of working with NP based therapeutics is the flexibility in controlling/modifying its physiochemical properties thus controlling the consequence of protein nanoparticle interactions. Hence in the thesis, we synthesized ZnONP to explore the effect of varying size, surface charge and hydrophobicity on the conformational dynamics of different proteins with varying aggregation prone region (APRs). In the beginning, we have reported the highly aggregation prone nature of recombinant hGPx7 and shown that the ZnONP does not show any significant effect on its aggregation propensity. With the help of computational tools, we also identified some exposed hydrophobic APRs in close proximity with its protein binding sites, indicating the role of protein-protein interaction in maintaining the compact stable state of hGPx7. The next part of the thesis gives an insight about the role of solvent polarity in disturbing protein stability and the chaperone like behaviour of ZnONP under SDS induced fibrillation of lysozyme at pH 9.0. The competitive binding between SDS and ZnONP on the exposed hydrophobic patch of lysozyme in pH 9.0 solution, was mediated mainly through electrostatic and hydrophobic interactions. The preferential binding of the lysozyme onto ZnONP inhibits the protein fibrillation by enhancing its secondary structure and activity. However, the same ZnONPs showed contrasting effects on insulin, a small globular protein vital for glucose regulation and its aggregation has been implicated in type II diabetes. However, surface functionalization of ZnONP with tryptophan and tyrosine, not only mitigates the fibrillation of insulin induced at bare ZnONP interfaces but also reduced the toxicity of oligomers formed in their presence. After exploring the effects of ZnONP on globular protein, our last choice as protein was an intrinsically disordered protein, α-synuclein. We investigated the effects of ZnONP and its varying size/surface curvature on the fibrillation propensity of α-synuclein. A concentration as well as size dependent inhibition in α-synuclein fibrillation was observed. ZnONP stabilizes the native structure of synuclein and inhibit the synuclein fibrillation by preferably binding to hydrophobic cluster of α-synuclein monomers resulting in formation of off pathway non-toxic aggregates with native random coil content. Hence, exploring the effect of ZnONP on different proteins will contribute towards a better understanding of the aggregation processes, and may open the way to designing nanoparticles that modulate protein aggregation and formation of toxic amyloid species.