Using gold nanoparticles to assess protein accessibility and protein-protein interactions in complex networks

Geometric and structural features of protein networks are difficult to assess when networks lack a defined structure and when solubility of the involved proteins is limited. Solubility issues prevent using spectroscopic approaches, and amorphous structures make classical/advanced microscopic approaches almost inefficacious. We took advantage of the high thiol reactivity of the surface of Au nanoparticles (AuNPs) to test accessibility of protein thiols in a complex model network, involving water-insoluble gliadins and glutenins in durum wheat semolina, where proteins interact through both hydrophobic interactions and disulfide bonds. After removal of non covalently bound proteins, proteins bound to the AuNPs (either directly or through "piggybacking" on other proteins) were identified by MS/MS. No gluten proteins were bound to AuNPs (20 nm average size) in the absence of pre-treatments (low molarity urea or detergents) that loosened hydrophobic interactions in the grain proteins network. Gluten proteins bound in network-loosening conditions included both glutenins (containing both free cysteines and intramolecular disulfides), but also gliadins (having no free cysteines and many intramolecular disulfides), proving that the two protein classes are interacting in the grain through intermolecular disulfides. These preliminary results pave the way to using AuNPs of different size for testing the nature of interacting proteins, the chemistry of their interaction, and the geometrical features of the resulting network whenever cysteine-containing proteins are involved. This is obviously relevant to many food-related systems, but may be useful also for addressing the chemical and geometrical features of several pathologically relevant protein aggregates.