Data di Pubblicazione:
2012
Citazione:
PROTEIN UNFOLDING ON INTERFACES: A STRUCTURAL AND FUNCTIONAL STUDY / M. Miriani ; tutor: F. Bonomi. DIPARTIMENTO DI SCIENZE MOLECOLARI AGROALIMENTARI, 2012 Feb 10. 24. ciclo, Anno Accademico 2011. [10.13130/miriani-matteo_phd2012-02-10].
Abstract:
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 “gain of function” or the “loss of function” 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 -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’ 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’ 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’s tryptophans containing regions are exposed to the aqueous phase. However, the multiplicity of glycinin’s 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 “lock” the protein structure in a conformation that is insensitive to further phy
Tipologia IRIS:
Tesi di dottorato
Keywords:
protein ; unfolding ; hydrophobic interfaces
Elenco autori:
M. Miriani
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