The mechanism of enzyme regulation through conformational changes is a key pattern in governing cell behavior. In this thesis the focus is on three protein complexes that reflect how protein activity can be regulated by different effectors. Different spectroscopic techniques, like IR and Raman spectroscopy, were used is order to follow the secondary and tertiary conformational changes in protein structure to identify their roles. The first protein of interest was PDZ1 from MAGI-1, involved in cellular signaling. This scaffold domain is known to interact with the E6 protein from HPV16. It was demonstrated that the different conformational states and their affinities to the C-terminus of the viral protein is regulated by the dynamics of the hydrogen bonding network formed by the connection of specific amino acids in three regions of the protein. Study of mutations around the C-terminal area of the protein and the βC strand were performed; demonstrating that both regions are crucial for assembly of the hydrogen bonding network to stabilize the substrate binding. These results leads to conclude that the pathogenicity and prevalence of a particular virus like HPV16 is in its ability to build a stronger hydrogen bonding network in comparison to the natural binder. The allosteric model and the "shift population" model agree that, upon binding, conformational changes distant from a carboxylate binding group might be the key to understanding the binding dynamics between the PDZ domains and the viral proteins.The second protein of interest was a model that constitutes a small scale prototype of the conformational changes observed in more complex proteins; it is a short Copper-binding peptide, the amyloid-beta peptide, known to beinvolved in Alzheimer's disease. The objective with this model was to describe the effect of histidine ligands in the metal centers upon Copper (Cu) reduction, a key electrochemical reaction in the development of Alzheimer's. FTIR difference spectroscopy showed two different spheres of coordination for Cu(II) and Cu(I). The major changes in the structure are dominated by the contribution of the imidazole ring of His residues (His6, His13 and His14), in addition to Asp1 and Tyr10 residues. Changes in the coordination geometry could be key to the pH-dependency of the aggregation observed in the presence of Cu(I). Accordingly, it can be suggested that the formation of the fibrils observed in Alzheimer's patients is not only triggered by the presence of Cu but it is strongly affected by its redox state. The last system of interest was a metalloprotein, the NADH:ubiquinone oxidoreductase (complex I), which plays a key role in the cellular bioenergetics. This protein bears several Fe-S clusters and one flavin and its activity is regulated by the energy produced by a bound substrate and the electron transfer of its cofactors. The metal ligand-vibrations of the cofactors are described in their oxidized and reduced states. Using electrochemistry coupled to FTIR, Resonance Raman and Fluorescence spectroscopies, the investigation of complex I led to the conclusion that the properties of the metal centers are dictated, to a large extent, by their surrounding environment. [...]
| Identifer | oai:union.ndltd.org:CCSD/oai:tel.archives-ouvertes.fr:tel-01048738 |
| Date | 24 April 2014 |
| Creators | Yegres, Michelle |
| Publisher | Université de Strasbourg |
| Source Sets | CCSD theses-EN-ligne, France |
| Language | English |
| Detected Language | English |
| Type | PhD thesis |
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