The investigation of structural aspects of wheat proteins and of their interactions with other components of the system is far from being conclusive because of the intrinsically disordered structure of the involved proteins, of the multiplicity of their interactions, and of methodological issues. In this work, we took advantage of a novel methodological approach to gather information on the geometry of the network formed by proteins in wheat semolina and on the nature of the interacting proteins. This approach is based on the high reactivity of Au+ ions on the surface of gold nanoparticles (AuNPs) towards thiols that results in the fast formation of covalent bonds between proteins and AuNPs, reportedly resistant to disulfide reductants commonly used in protein chemistry. Suspensions of wheat semolina were prepared in buffers allowing different degrees of swelling/relaxation of the protein network, and incubated with AuNPs with a nominal diameter of 20 nm. Protein-loaded AuNPs were recovered by simple centrifugation procedures and extensively washed under conditions favoring the breakdown of non-covalent interactions. Proteins covalently bound on the surface of AuNPs were identified by MS analysis of their proteolytic fragments. Gluten-forming proteins were found to be band to AuNPs only when detergents or chaotropes were added to the semolina/AuNPs suspension at room temperature. AuNPs-bound proteins also included gluten-forming proteins with no reported free thiols, suggesting that they may be piggybacked on other thiol-containing gluten-forming proteins via disulfide bonds already present in the untreated semolina. The potential of this approach may be relevant in studies aimed at exploring the geometry of thiols-containing protein networks, the nature of the involved proteins and of the interactions they establish in foods - and also in systems relevant to human health - as well as any modifications of these features upon processing.
Structural and topological insights of gluten-forming proteins by their covalent binding to gold nanoparticles
M. Marengo;
2019-01-01
Abstract
The investigation of structural aspects of wheat proteins and of their interactions with other components of the system is far from being conclusive because of the intrinsically disordered structure of the involved proteins, of the multiplicity of their interactions, and of methodological issues. In this work, we took advantage of a novel methodological approach to gather information on the geometry of the network formed by proteins in wheat semolina and on the nature of the interacting proteins. This approach is based on the high reactivity of Au+ ions on the surface of gold nanoparticles (AuNPs) towards thiols that results in the fast formation of covalent bonds between proteins and AuNPs, reportedly resistant to disulfide reductants commonly used in protein chemistry. Suspensions of wheat semolina were prepared in buffers allowing different degrees of swelling/relaxation of the protein network, and incubated with AuNPs with a nominal diameter of 20 nm. Protein-loaded AuNPs were recovered by simple centrifugation procedures and extensively washed under conditions favoring the breakdown of non-covalent interactions. Proteins covalently bound on the surface of AuNPs were identified by MS analysis of their proteolytic fragments. Gluten-forming proteins were found to be band to AuNPs only when detergents or chaotropes were added to the semolina/AuNPs suspension at room temperature. AuNPs-bound proteins also included gluten-forming proteins with no reported free thiols, suggesting that they may be piggybacked on other thiol-containing gluten-forming proteins via disulfide bonds already present in the untreated semolina. The potential of this approach may be relevant in studies aimed at exploring the geometry of thiols-containing protein networks, the nature of the involved proteins and of the interactions they establish in foods - and also in systems relevant to human health - as well as any modifications of these features upon processing.
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