Assessing the geometrical features of protein-protein interactions in gluten

This study exploits the reactivity of the surface of Au nanoparticles (AuNPs) towards thiols to test accessibility of protein thiols in protein in durum wheat semolina, where proteins interact through both hydrophobic interactions and disulfide bonds. After removal of loosely bound proteins, proteins covalently bound the AuNPs were identified by MS/MS after limited proteolysis. We found it necessary to loosen hydrophobic interactions in the network to grant accessibility to AuNPs (20 nm average size). This required the use of low molarity urea or detergents, and the use of appropriate controls to verify binding of AuNPs to proteins that may be solubilized under the same mild-denaturing conditions. Both glutenins (containing free cysteines and intramolecular disulfides) and gliadins (having no free thiols and only intramolecular disulfides) were found to be covalently bound to AuNPs, proving that the two protein classes are interacting in the grain through intermolecular disulfides. Current tests are aimed at verifying how these results can be affected by previous blockage of some of the accessible free thiols on these proteins prior to exposure to AuNPs, and are carried out by using specific fluorescent probes. This combination of approaches should provide further insights into the nature of the involved proteins and the geometry of their interactions.