Étude d’interactions biologiques à l’aide de la résonance des plasmons de surface et de la spectroscopie de fluorescence
The understanding of biological interactions is of utmost importance if we are to comprehend the complex mechanisms taking place in living beings. These interactions can involve proteins, oligonucleotides and peptides, but are not limited to these types of biomolecules. Biological interactions are characterized by their affinity and binding kinetics. Therefore, it is extremely important to have an analytical tool able to measure these properties in order to characterize the interaction between biological molecules. This Master’s research had the objective to optimize surface plasmon resonance spectroscopy (SPR) to study biological interactions of poorly characterized biomolecular interactions and to measure both affinity and kinetic parameters of the binding between several sets of relevant biological molecules. The SPR technique relies on the immobilization of a biological receptor on a gold-coated prism to create a biosensor. One of the studied biomolecules is anchored on the surface while the other one is flowed through a fluidic cell placed on top of the prism. The resulting measured signal results from the binding between the two biomolecules and allows us to determine thermodynamic (KD) and kinetic (kon and koff) parameters associated with the biological complex. These properties of SPR were exploited in this thesis to study the interaction between the protein CD36 (Cluster of Differentiation 36) and peptides homologous to the GHRP-6 (Growth Hormone Releasing Peptide 6), the interaction between a ganglioside (GM1) and the Cholera toxin B sub-unit (CTB) and a synthetic peptide, the interaction of cytokines and the proteinaceous soluble receptor EBI3 (Epstein-Barr virus Induced Gene 3) and the interaction between GFP (Green Fluorescent Protein) and another protein provided by a collaborator. The sensors developed for this Master’s project allowed us to study complex interactions for which few standard methods of analysis exist. This Master’s thesis also briefly describes an instrument prototype able to monitor either bulk or surface fluorescence during the SPR assay.
Fluorescence Surface plasmon resonance Biological interactions Affinity Kinetic