Elucidating the structural changes of bovine betalactoglobuline after adsorption on a hydrophobic nanostructured surface

The spontaneous adsorption of protein molecules on interfaces is a ubiquitous phenomenon in natural and man-made systems. This phenomenon plays a central role in many fields, such as health, food, environmental science, and biochemical or immunochemical analysis. The structural rearrangement caused by the direct contact with the sorbent phase may affect protein biological activity, including allergenicity, bioavailability, and ability to bind micro- and macromolecular ligands. Whether proteins unfold randomly or through subsequent ordered and eventually reversible steps remains often unknown, and information about the molecular determinants of the “gain of function” or the “loss of function” observed upon adsorption is scarce. The aim of this work is to understand the structural changes that bovine beta-lactoglobulin (Blg) undergoes after adsorption on hydrophobic nanostructurated surfaces, in our case 46 nm polystyrene nanoparticles. Intrinsic fluorescence spectroscopy was used to evaluate tertiary structural changes, along with the binding of fluorescent dyes (ANS), and accessibility of reactive cysteine thiols. Limited proteolysis with trypsin was performed on both the adsorbed and the native protein. The intrinsic fluorescence spectrum of adsorbed Blg is red-shifted compared with the free-protein one thus indicating that the adsorbed protein assumes a new structure in which Trp19, usually buried inside the hydrophobic core, is exposed to water. Moreover, adsorbed Blg increases ≈2 folds its global quantum yield. This phenomenon could be explained either by the moving of Trp61 away from the Cys66-Cys160 disulphide bond, and/or by the moving of Trp19 from Arg124, thus removing fluorescence-quenching interactions within the protein structure. The only free thiol in Blg is on Cys121, which is buried in the native structure, but becomes readily and almost completely accessible after adsorption. The overall Blg surface hydrophobicity seems to increase after interaction with the hydrophobic surface, confirming the occurrence of major rearrangements. The adsorbed protein is resistant to proteolysis by trypsin where the free protein is almost completely digested in the same conditions. All these data demonstrate an extended stretch of the native structure after adsorption on hydrophobic surfaces with the exposure of new protein regions usually buried from the aqueous media. Future studies will be focused to assess whether adsorbed Blg is still able to bind its natural ligands (e.g. vitamin D), and to evaluate changes in its immunoreactivity (Blg is a major food allergen).