Paramagnetic NMR Spectroscopy Is a Tool to Address Reactivity, Structure, and Protein-Protein Interactions of Metalloproteins: The Case of Iron-Sulfur Proteins

The study of cellular machineries responsible of the Iron-Sulfur (Fe-S) cluster biogenesis has led to the identification of a large number of proteins, whose importance for life is documented by an increasing number of diseases linked to them. The labile nature of Fe-S clusters and the transient protein-protein interactions, occurring during the various steps of the maturation process, make their structural characterization in solution particularly difficult. Paramagnetic NMR has been used since decades to characterize chemical composition, magnetic coupling and electronic structure of Fe-S clusters in proteins; it represents therefore a powerful tool to study the protein-protein interaction networks of proteins involving into Iron-Ssulfur cluster biogenesis. The optimization of the various NMR experiments with respect to the hyperfine interaction will be summarized here in a form of a protocol; recently developed experiments for measuring longitudinal and transverse nuclear relaxation rates in highly paramagnetic systems will be also reviewed. Finally, we will address the use of extrinsic paramagnetic centers covalently bound to diamagnetic proteins, which contributed over the last twenty years to promote the applications of paramagnetic NMR well beyond the structural biology of metalloproteins