Multilayer protein corona on gold nanorod surface: First evidence of soft corona protein-protein interactions using solution NMR spectroscopy

The nanoparticle surfaces interact readily with biomolecules, including proteins forming a corona. The protein layer that directly interacts with the nanoparticle surface is usually explored. Non-covalent interactions of proteins in the second layer have not been elucidated. We report for the first-time multilayer protein-protein interactions in ubiquitin corona on gold nanorod surface. The conformational dynamics of the protein, ubiquitin, on the surface of gold nanorods were probed at the molecular level using two-dimensional solution nuclear magnetic resonance spectroscopy. The interaction of Ubiquitin with gold nanorods was characterized by UV–visible spectroscopy, dynamic light scattering, and zeta-potential measurements. The residues of Ubiquitin that show significant perturbations fall into two categories: (a) the residues that directly interact with the surface of gold nanorods forming hard corona (primary interaction) and (b) the residues that are forming soft corona (secondary interaction). The formation of hard corona on the surface of the nanorod takes place even at a low concentration of the protein. The protein does not undergo any changes in its secondary or tertiary structure. As the protein concentration is increased, the hard corona, which is off mutually interconvertible multiple conformers, undergoes conformational reorientation to maximize its interaction with the nanorod surface. The study reveals electrostatically driven weak protein-nanorod interactions. Further, the study reveals two different processes, one involving the direct interaction with nanorods and the other involving protein corona formation in the protein-nanomaterial system. The study, for the first time, explores multilayer protein corona on a nanoparticle surface and provides new insights into the mechanisms involving protein aggregation in the presence of the nanoparticles.