PROTEIN UNFOLDING ON INTERFACES: A STRUCTURAL AND FUNCTIONAL STUDY

The spontaneous adsorption of protein molecules on interfaces is an ubiquitous phenomenon in natural and man-made systems. The structural rearrangement caused by the direct contact with the sorbent phase may affect protein biological activity, including bioavailability, and ability to bind micro- and macromolecular ligands. Moreover, protein immunoreactivity has been assessed to change if protein molecules interact with an hydrophobic phase; indeed adjuvant are hydrophobic substances that act as enhancers in antibodies production. Whether proteins unfold randomly or through subsequent ordered and eventually reversible steps remains often unknown, and information about the molecular determinants of the \u201cgain of function\u201d or the \u201closs of function\u201d observed upon adsorption is scarce. The aim of this work is to understand the structural and functional changes that soy storage proteins (beta-conglycinin and glycinin) and bovine betalactoglobulin (BLG) undergo after adsorption on hydrophobic nanostructured surfaces. Protein conformational changes after adsorption on interfaces were evaluated by using different techniques, including fluorescence and solid-state fluorescence spectroscopy, CD spectroscopy, along with limited proteolysis followed by recognition of released peptides by MS. Moreover, changes in biological behavior were evaluated by measuring changes in immunoreacivity that may be relevant from the standpoint of immune response or immunomodulation. Experiment aimed to evaluate the influences of interface denaturated protein on live cells were carried out. For this purpose BLG and BLG-stabilized emulsions, both labeled with FITC, were incubated with monocyte and differences in protein uptake were evaluated by citofluorimetry. In order to have a model of BLG denaturation on the polystyrene interface, an in silico study was performed. The simulation was carried out using the computational suite MOE (Molecular Operating System). In this work structural changes of \uf062-conglycinin and glycinin in solution were compared to those occurring when the proteins are adsorbed at the oil-water interface. Both proteins undergo structural modifications after adsorption on the oil droplet surface. From the standpoint of protein chemistry, the modifications occurring at the interface with the proteins investigated here have some peculiar traits, in what both these proteins expose their tryptophan-containing extension regions to the aqueous phase rather than to the droplet interior, as observed for other proteins. It is very important to note that, in beta-conglycinin, tryptophans are present in the extension domains of alpha and alpha\u2019 subunits, and the present fluorescence data confirm previous results demonstrating that the polar extension regions in these proteins are important for their emulsifying ability. These results support the hypothesis that while the a and a\u2019 core domains interact with oil phase, the extension regions protrude into the aqueous phase and stabilize the emulsion droplets by providing the necessary polar regions. Also glycinin\u2019s tryptophans containing regions are exposed to the aqueous phase. However, the multiplicity of glycinin\u2019s genetic variants makes it much more challenging to derive definite answers from the hydrophobicity profiles of this protein, and some more detailed proteomic work is needed to better understand which portion of the protein anchors to the interface. It is also interesting to note that heat treatment does not affect the structural features of either protein once they are adsorbed at the oil-water interface. In other words, the modifications occurring upon adsorption at the interface appear to \u201clock\u201d the protein structure in a conformation that is insensitive to further physical denaturation, at least under the temperature/time regimes employed in this work. As a matter of fact, it is somewhat expected that, in emulsions, the structural regions more sensitive to the entropic changes ensuing from alteration in the water structure (i.e., the protein hydrophobic core) are at least partially buried into the non-polar lipid phase, and thus are insensitive to temperature-dependent changes in the colligative properties of the solvent. The various peculiarities of these systems and their practical relevance seem worth further investigation. We are currently addressing the molecular details of the observed events, in an attempt to identify specific molecular determinants of the different behaviour of these proteins, as well as the changes occurring during heating, and to assess whether the conformational changes reported here result in biologically relevant modifications when emulsions are consumed as food. Also BLG structure changes after interaction with an hydrophobic interface. 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 482 folds its global quantum yield. 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. BLG sensitivity towards trypsin \u2013 and therefore the resulting peptidic pattern - is modified as a function of the hydrophobic support where the protein is adsorbed. In fact, in the case of NP-adsorbed BLG trypsin resistance is similar to the one of free BLG, whereas it dramatically decreases for emulsion-BLG. 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. Changes in immunoreactivity occurred after adsorption on hydrophobic surfaces. BLG adsorbed on oil droplet surface is more reactive ( 4835%) than the free protein by using the 5G6 MAB, and also it is more reactive ( 48110%) when using the 1E3 MAB. BLG adsorbed on latex NP is likely to increase its immunoreactivity by using both MAB, that indicates that BLG assumes different structures as a function of the interacting interface(s). Cells experiments show how the BLG internalization by monocites follows two different kinetics according to protein physical state. Moreover the absorption of adsorbed BLG seems to be not influenced by competition of free BLG, leading us to hypothesize the presence of two different pathway for the protein internalization depending on their physicals state. The in silico denaturation simulations demonstrate that the interaction orientation is fundamental for the type and magnitude of protein structure reorganization. The system, all build by us, seems to be very stable, and the latex denaturating interface should be used with others proteins. In conclusion in this thesis I described in deeply the structural modification that three protein, whit a huge importance for nutrition and food science, undergo after adsorption on different model hydrophobic interfaces. I also produced an in silico model for computational prediction of protein denaturation on polystyrene interface. Physiological implication regarding protein structural reorganization were also explored