Structure chemical shifts restraints

Fig. 2. Structure determination by NMR. Labelled samples are prepared by chemical or biochemical means. Application of multidimensional NMR pulse sequences delivers the assignment of the resonances. By measuring H,H distances from NOE or ROE, J couplings, cross-correlated relaxation rates, dipolar couplings, and chemical shifts, restraints are derived that can be used for structure calculation.

Structure calculation algorithms in general assume that the experimental list of restraints is completely free of errors. This is usually true only in the final stages of a structure calculation, when all errors (e.g., in the assignment of chemical shifts or NOEs) have been identified, often in a laborious iterative process. Many effects can produce inconsistent or incorrect restraints, e.g., artifact peaks, imprecise peak positions, and insufficient error bounds to correct for spin diffusion. 

As another example, the three-dimensional structure of Cytochrome c has been determined on the basis of structural information from pseudocontact paramagnetic chemical shifts, Curie-Dipolar cross-correlation, secondary structure constraints, dipolar couplings and 15N relaxation data [103]. This protein has a paramagnetic center, and therefore the above-mentioned conformational restraints can be derived from this feature. Dipolar couplings do not average to zero because of the susceptibility tensor anisotropy of the protein. The structure determination of this protein without NOE data gives an RMSD (root... 

In Fig. 1 we have highlighted with a dark background the different types of NMR methods that are used in drug-discovery projects. These include basic ID and 2D methods that are used to confirm the identity of peptides, determine their conformation, or derive restraint information used in 3D structure calculations (left side of Fig. 1). Methods to study binding interactions (middle section of Fig. 1) can be broadly categorized as being based on NOEs, diffusion, relaxation, or chemical shift changes. NOE-based methods include the transferred NOE... 

The main source of conformational information for biopolymers are the easy-to-obtain chemical shifts that can be translated into dihedral restraints. In addition, for fully 13C labeled compounds, proton-driven spin diffusion between carbons [72] can be used to measure quantitatively distances between carbons. The CHHC experiment is the equivalent of the NOESY in solution that measures distances between protons by detecting the resonances of the attached carbons. While both techniques, proton-driven spin diffusion and CHHC experiment [73], allow for some variation in the distance as determined from cross-peak integrals, REDOR [74] experiments in selective labeled compounds measure very accurate distances by direct observation of the oscillation of a signal by the dipolar coupling. While the latter technique provides very accurate distances, it provides only one piece of information per sample. Therefore, the more powerful techniques proton-driven spin diffusion and CHHC have taken over when it comes to structure determination by ss-NMR of fully labeled ligands. 

The nondegenerate geminal pairs are usually named according to their chemical shifts (e.g., downfield of ft ) rather than their stereochemical relationships (pro-R and pro-S). In structure calculations, this usually is dealt with by creating a pseudo-atom right between the pro-R and pro-S positions in 3D space. The NOE restraints are applied to the pseudoatom and not to the real atoms, and the distance limit is increased a bit to account for the ambiguity (we do not really know which restraint applies to which of the two positions in space). Similarly, a pseudoatom is created at the center of the three equivalent protons of a CH3 group, and the distance restraint is applied to the pseudoatom. 

There are three aspects to consider. First, we summarize briefly the underlying computational framework needed and the general strategy used in the structure determination. Second, we cover the use of 2D, 3D, and 4D methods to permit the sequential assignment of peaks to specific amino acids. Finally, we describe the use of nuclear Overhauser enhancements and spin coupling constants to provide restraints on interproton distances and bond angles, and we indicate how dipolar coupling and chemical shifts can sometimes add further information on molecular conformation. 

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