Protein structure and dynamics from paramagnetic and diamagnetic relaxation in solids
Résumé
We will present site-specific measurements of 15N and 13C relaxation rates, for crystalline superoxide dismutase (SOD). By combining high magnetic fields, fast MAS, and extensive deuteration, it is possible to acquire quick and resolved 1H,15N fingerprints of the molecule, which can be conveniently edited in order to measure 15N or 13C relaxation rates. In this approach, more than 100 relaxation curves were obtained in only few days on less than 4 mg of protein. Sizable PRE were measured for 15N and 13C nuclei between 12 and 24 Å from the Cu ion, and were applied as structural restraints, in addition to 1H-1H short-range distance restraints and chemical shift-based dihedral restraints. The inclusion of PREs reduces the RMSD for the structure bundle by 50%. Additionally, nuclear relaxation rates provide a powerful probe of protein dynamics. We have recently shown how to measure a complete set of rates for a site-specific motional determination and here we have measured the 15N R1r and 15N R1 measurements, as well as 15N R1r to characterize the conformational selection events in the active site associated with copper binding in SOD.