Aplicación de la resonancia magnética nuclear de sistemas paramagnéticos a la determinación de propiedades estructurales y dinámicas de las proteínas rusticianina y calbindina DqK

Zusammenfassung

Nuclear Magnetic Resonance (NMR) is the only methodology that allows the procurement of detailed, high quality structural information of biomolecules in solution at atomic level. NMR permits, as well, the complete dynamic characterization of a protein, and the acquaintance of its hydration properties. When working with NMR, substantial advantages can be derived from the existence of paramagnetic ions. The paramagnetism provides essential structural information not only from the metal site surroundings, but also from the whole protein. This PhD Thesis represents an exhaustive example of this last statement. Two different and complementary aims were pursued and reached in this Thesis. The first one was methodological. We apply NMR to paramagnetic systems in order to obtain information on the electronic properties of the metal, on the structure of the whole systems, and also on its dynamic features. The second one was strictly biochemical. We have characterised the blue copper protein rusticyanin (Rc) and the calcium binding protein calbindin D9k (Cb) by paramagnetic NMR and we have correlated structural and dynamic aspects of each protein with their functional properties. We have characterized the metal-ligand interplay in Rc by paramagnetic NMR studies on the copper(II) native protein, the cobalt(II) derivative, and also on five different mutants. The electronic structure of the copper ion is defined by the orientation of the histidine ligands, while the interaction of the copper ion with the cysteine and the methionine ligands is independently determined by the protein folding around the metal site. We have also stated in the present Thesis that the high redox potential of Rc is due to the high hydrophobic environment of the copper ion. Studying dynamic and hydration features of Rc by 1H-15N NMR we can assert that it is the most rigid and hydrophobic BCP. We have selected monocerium(III) substituted Cb to study the relative importance of paramagnetism-based constraints with respect to classical ones in solution structure determinations of paramagnetic metalloproteins. The adequate use of these constraints gave us a very refined structure(RMSD=0.25 Å). We have also monitored the unfolding of the calbindin D9k by 1H and heteronuclear NMR. The hydrophobic core of the protein shows a tighter packing at GdmHCl 2 M. This is one of the main factors for the great stability of the protein.