Paramagnetic NMR restraints for the characterization of protein structural rearrangements

Conformational rearrangements in proteins are often key steps of functional regulation. These rearrangements often require the system to visit high-energy (i.e.: transient and scarcely populated) states. The characterization of such transient states can rely only upon very few methodologies. Among those, we can count NMR, which is an outstanding tool for the atomic-resolution characterization of dynamic processes in biomolecules. Even more so, when aided by the use of paramagnetism-based restraints, like paramagnetic relaxation enhancements (PREs), pseudocontact shifts (PCSs) and paramagnetic residual dipolar couplings (RDCs) [1], NMR turns out to be very informative to shed light on protein dynamics. Paramagnetic effects are very sensitive to even modest structural rearrangements, not observable through the more conventional (e.g. NOE) detection [2]. These NMR data can be measured in metalloproteins by replacing the diamagnetic metal ion with a paramagnetic ion (or vice versa), switching between paramagnetic and diamagnetic states of the same metal, and in all biomolecules upon attachment of a paramagnetic tag [3, 4, 5, 6, 7]. They are in fact obtained from the difference in relaxation rates (PREs), NMR shifts (PCSs), and 1J-couplings (RDCs) between the paramagnetic and the diamagnetic forms of the protein [8]. When paramagnetic tags are used, it is very important that these tags are rigidly attached to the proteins, because tag mobility can largely reduce the possibility of retrieving information on the occurring protein mobility.

The potentialities of paramagnetic NMR data to shed light on conformational rearrangements can be even more profitably exploited when complemented by other experimental techniques, including X-ray crystallography and cryo-electron microscopy, or by computational studies [9]. Single-molecule Förster resonance energy transfer (FRET) [10], small-angle X-ray scattering (SAXS) and double electron–electron resonance (DEER) [11] can also result useful complementary techniques.

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