This method has been principally used for the characterization of protein-carbohydrate interactions, after its introduction by Meyer group (Mayer and Meyer, 1999). Thus, it was used to resolve the binding substrate specifity of yeast hexokinase PII (Blume et al., 2009) and to resolve the hydrogen atoms of xylobiose involved in sugar-protein interaction. H2-5 of xylobiose were identified as critical non-covalent interactions in wild type GH10 xylanases, which were absent in Epigenetic inhibitor the

E159Q mutant, indicating the importance of negative charge in the substrate binding (Balazs et al., 2013). Another important application of this method has been in drug discovery (Bhunia et al., 2012). In the late 1950s the PRE theory for static systems was established and since then it has been used in characterizing paramagnetic metalloproteins Dwek (1973). One application was to measure

the relaxation of water by paramagnetic metals in the presence of enzymes and its substrates to determine the coordination of the metal PD0325901 at the active site of the complex substrate–enzyme. It is a rapid and sensitive method for measure ligand–enzyme interaction, where the enzyme system is appropriate, to measure the effect of ligand binding on the solvent (1H of H20). This method requires a paramagnetic probe that can affect the longitudinal relaxation rate of the solvent. The probe elicits an effect on the proton longitudinal relaxation rate (PRR or PRE) to give a proton relaxation rate Amino acid enhancement. If the enhancement effects are sensitive to ligand binding, then studying the environment around the probe can yield important thermodynamic and structural information of the

complexes formed among the enzyme, substrates and the metal. Although a number of probes can be used for these studies, Mn(II) has been the most frequently used due to its physical–chemical properties and its usefulness in many cases. To determine protein structure, this method has had a new impulse with the introduction of biochemical methods to label proteins with paramagnetic probes at specific sites and the development of appropriate computational methods (Donaldson et al., 2001). However the most interesting application of this method has been the detection of transient low population species that remain in rapid exchange with the major specie that modulates the transverse PRE observed (Clore, 2011). In addition to structures and ligand binding thermodynamics, nuclear magnetic resonance can yield information on the dynamics of the structural regions of the protein. This usually involves measuring relaxation times such as T1 and T2 to determine order parameters (S2), correlation times, and chemical exchange rates. NMR relaxation is a consequence of local fluctuating magnetic fields within a molecule. Molecular motions generate local fluctuating magnetic fields.