Our laboratory is equipped with a Biacore X100, Surface Plasmon Resonance apparatus (SPR), a system using the SPR phenomenon to monitor in real time and in a label free environment biomolecular interactions occurring between a ligand immobilized on a sensor chip surface and an analyte in solution.

The association between molecules is revealed only indirectly by monitoring variation of the refractive index near the sensor surface with an optical method. In a SPR experimental set-up, incident light is reflected from the internal face of a prism in which the external face has been coated with a thin metal film. In condition of total internal reflection the reflected photons create an electric field (evanescent field) on the opposite side of the interface. At a particular critical angle (resonance angle), the evanescent wave resonates with the oscillation in the electrons (plasmons) on the surface of the metal film (hence the term “Surface Plasmon Resonance”) and this causes a drop in the reflected light. Since the critical angle is dependent on the refractive index of the medium present on the metal surface, this real-time method is used to measure molecular interactions at solid-liquid interface between a ligand immobilized to the sensor surface and the analyte in solution. When the ligand and the analyte interact to form a complex, this causes a change in the refractive index and thus a shift of the resonance angle that is monitored in real time and reported in a graph called sensorgram.

The technique, simple and non-invasive, provides unique and global data on the interactions between biomolecules: binding/non-bonding, selectivity of binding, affinity, active concentration and kinetics of binding. In addition, availability of a wide range of sensor chips for our apparatus Biacore X100, allows to analyze most of the molecular interactions (proteins, nucleic acids, lipids, carbohydrates, cells).

In the context of anticancer drug research on which we are interested, we have used SPR to study the ternary interaction between Azurin, p53 and Mdm2 (Domenici et al., 2011).

The bacterial protein Azurin has been shown to inhibit cancer cell proliferation by binding and stabilizing p53 through the reduction of its Mdm2-mediated proteasomal degradation. To investigate if and how Azurin interferes with the Mdm2-p53 association, a detailed characterization of the binding interaction of these three proteins has been performed. Very interestingly we have found that the three proteins are likely engaged in a ternary interaction (as also suggested by the work of Funari et al. in which the AFS technique has been used) whose kinetics points out that binding of Azurin to p53 causes a significant decrease of the Mdm2-p53 association rate constant and binding affinity, without hindering the accessibility of Mdm2 to the binding pocket of p53. Hence, we have significantly contributed in understanding the azurin mode of action by demonstrating that the bacterial protein stabilizes p53 through a non competitive inhibition of the Mdm2-p53 interaction but, perhaps, by an allosteric mechanism.