In some cases where one nucleus of a ligand is very close to the paramagnetic center compared to other nuclei measured, the relaxation may be so efficient
that the nucleus may be in slow exchange (T2M⪡τM) (1/fT2p=1/τM). If this is the case, then a temperature-dependence of 1/fT2p will give a value for koff and for the energy of activation, Ea, for the ligand exchange process. In this case the structure of the ligand at the catalytic site (from 1/T1M), its exchange rate, and the energy barrier for this exchange process, can be obtained and compared with these parameters for the unmodified enzyme. In the case where the exchange process is simple, and Kd (=koff/kon) for ligand binding is known, the value of kon, can also be estimated ( Monasterio, 1987 and Monasterio, 2001). Knowledge buy Vorinostat of the three-dimensional structure of a polypeptide or protein (enzyme) is a prerequisite to the understanding of its physical, chemical, and biological properties. Since the time that Perutz and Kendrew determined the structure of hemoglobin and myoglobin, more than five decades ago,
about 750 non-identical structures of a total number of 270 have been determined by crystallographic BIBW2992 supplier and NMR techniques (Orengo, 1994). The precision with which the NMR structures of small proteins can now be determined approaches that of moderately good X-ray crystal structures. In the protein interior, the structures obtained from the highest quality NMR data can be as precise as all but the very best X-ray structure, whereas the surface residues often appear disordered in solution and hence in the NMR structures derived from solution data. Thus, the main differences between the NMR and X-ray
structures of proteins are in fact usually found on protein surfaces. In the last few years the significant increase in the number of known three-dimensional structures of small proteins in solution became possible due to advances in NMR technology such as the development of superconducting magnets, Fourier transform spectroscopy, computer control of the instrumentation and new multidimensional NMR techniques developed by Ernst (Ernst et al., 1987), who won the Nobel Prize in 1991. The basic find more steps for protein determination from NMR are the following: (1) Assignment of resonances signals to individual nucleus. (2) Determination of distance constrains and dihedral angle constrains from NOE׳s and J couplings, respectively. (3) Calculation of a family of three-dimensional structures on the basis of the distance restrains, supplemented if possible by some torsion-angle restrains derived from coupling constants. (4) Refining of the structures by using geometric constrains and potential energy functions, for instance, with restrained energy minimization and restrained molecular dynamics. These steps will be discussed in some detail.