Structure determination of biological macromolecules, such as proteins and nucleic acids, is crucial to understanding their biological function. NMR spectra provide a wealth of information on molecular structure. In addition, NMR spectroscopy facilitates studies of conformational changes induced by ligand binding in protein-ligand, protein-DNA, and protein-protein interactions, providing insights into structural biology. This information is pivotal for modern knowledge-based drug design and protein engineering, and for their applications in biotechnology.
The three-dimensional structure of the molecule, in terms of interatomic distances and angular geometries, is present in NMR spectra through different kinds of restraints, all sensitive to local bonded geometry or orientation of the molecule. Distance restraints, i.e. information on the proximity of particular nuclei, are determined from NOE/ROE intensities, which are inversely proportional to interatomic distances. Dihedral restraints are extracted from scalar couplings to supply information on torsional angles. Chemical shift restraints, which are readily available after assignment of NMR signals, enable loose definition of backbone dihedral angles φ and ψ. Projection restraints, which define the orientations of different interatomic vectors with respect to each other, can be used as long-range structural restraints. These can be measured, for example, from cross-correlated relaxation rates (Reif et al. 1997), from diffusion anisotropy by measuring T1/T2 ratios for 15N and 13C (Tjandra et al. 1997), or from residual dipolar couplings (Tolman et al. 1995). In addition, recently measured J-couplings across hydrogen bonds have become valuable constraints for structure determination (Dingley & Grzesiek 1998).
The present thesis focuses on different NMR methods devised for measurement of scalar and residual dipolar couplings in the protein backbone. Scalar couplings (or J-couplings) provide important structural information as they relate to dihedral angles via Karplus equations (Karplus 1959; Bystrov 1976), and their magnitude and sign reflect the local bonded geometry. There are three torsion angles in the polypeptide chain, which can be determined by NMR spectroscopy. The most important of these is the φ angle, which defines the dihedral between intraresidue amide and alpha protons. The size of this coupling constant is usually indicative of whether the corresponding amino acid residue resides in the α-helical or β-sheet region (Wüthrich 1986). Scalar coupling information is very useful in protein structure refinement as a structural constraint for defining the angle between two bond vectors, e.g. N-H and C-H.
Dipolar couplings have been used for structure determination in small molecules since the 1960’s (Saupe & Englert 1963), but their applicability for protein structure determination was demonstrated only very recently (Tolman et al. 1995; Tjandra et al. 1996; Tjandra & Bax 1997). The information content in dipolar couplings is quite different to that obtained from scalar interactions. Unlike scalar interaction, dipolar coupling is not a molecular constant. It originates from the through-space magnetic interaction between nuclei. Thus, dipolar interactions depend on interatomic distances and on the orientation of the internuclear vector with respect to the applied magnetic field (Saupe & Englert 1963).
In high-resolution NMR, dipolar couplings average to zero in solution, owing to the isotropic tumbling of molecules. However, minute residual dipolar couplings can be measured from macromolecules due to their intrinsic magnetic susceptibility anisotropy, and consequently, their small degree of alignment with the magnetic field (Tolman et al. 1995). A larger alignment emerges by dissolving the molecule in an anisotropic medium (Bax & Tjandra 1997). This enables a more reliable measurement of residual dipolar couplings.