Unraveling the mechanism of graphene oxide-mediated disruption of protein dimers

Functional graphene-derivatives have garnered significant interest in biomedical research due to their unique structural and physicochemical properties. However, poor understanding of their interactions with the key biomolecules, such as proteins, constrained their application. The cytotoxicity of graphene materials has often been linked to their effect on impairing protein-protein interactions. The mechanistic aspects of the disruption of protein-protein interactions are largely unknown. Herein, we investigated the mechanistic insights into the conformational changes of β-lactoglobulin (β-LG) protein induced by graphene oxide (GO) using spectroscopic, microscopic, and molecular dynamics (MD) techniques. Our experimental studies reveal a distinct interaction pattern between β-LG dimers and monomers with GO, indicating that GO disrupts the intermolecular forces of dimeric β-LG, dissociating it into monomers, as confirmed by secondary structure analysis and native-PAGE. Furthermore, morphological changes in the GO-protein complex were analyzed using electron, force, and fluorescence-based microscopy. The energy profile by umbrella sampling (US) simulations reveals monomer adsorption onto GO (-8.2 kcal mol^-1) is thermodynamically more favourable than dimer adsorption (-5.0 kcal mol^-1), which clearly depicts GO's ability to destabilize dimeric forms, facilitating monomer dissociation and adsorption. This disruption is linked to the heterogeneous surface of GO, where sp² regions enable π-π stacking with hydrophobic residues, and sp³ regions with oxygen groups form hydrogen bonds and interact with polar residues of β-LG. This study combines experimental and computational approaches to offer a comprehensive understanding of the role of GO on biomolecules, contributing to the broader evaluation of nanomaterial effects in biological systems.