Three-dimensional NOESY spectroscopy

Ikura M, Kay LE, Tsudin R, Bax A. Three-dimensional NOESY-HMQC spectroscopy of a 13C-labelled protein. J Magn Reson 1990 86 204-209. 

NOESY NMR spectroscopy is a homonuclear two-dimensional experiment that identifies proton nuclei that are close to each other in space. If one has already identified proton resonances in one-dimensional NMR spectroscopy or by other methods, it is then possible to determine three dimensional structure through NOESY. For instance, it is possible to determine how large molecules such as proteins fold themselves in three-dimensional space using the NOESY technique. The solution structures thus determined can be compared with solid-state information on the same protein obtained from X-ray crystallographic studies. The pulse sequence for a simple NOESY experiment is shown in Figure 3.23 as adapted from Figure 8.12 of reference 19. 

In de novo three-dimensional structure determinations of proteins in solution by NMR spectroscopy, the key conformational data are upper distance limits derived from nuclear Overhauser effects (NOEs) [11, 14]. In order to extract distance constraints from a NOESY spectrum, its cross peaks have to be assigned, i.e. the pairs of hydrogen atoms that give rise to cross peaks have to be identified. The basis for the NOESY assignment... 

NMR experiments include COSY, TOCSY, Cheteronuclear NMR experiments, NOESY (nuclear overhauser enhancement spectroscopy) and ROESY (rotating frame overhauser effect spectroscopy) as well as other two- and three-dimensional methodologies (Fossen and Andersen, 2006). 

Multidimensional NMR spectra are not restricted to cases where the separate frequency axes encode signals from different nuclear types. Indeed, much of the early work on the development of 2D NMR was performed on cases where both axes involved chemipal shifts. The main value in such spectra comes from the information content in cross peaks between pairs of protons. In COSY-type spectra (COSY = Correlation SpectroscopY) cross peaks occur only between protons that are scalar coupled (i.e., within 2 or 3 bonds) to each other, whereas in NOESY (NOE Spectroscopy) spectra cross peaks occur for protons that are physically close in space (<5 A apart). A combination of these two types of 2D spectra may be used to assign the NMR signals of small proteins and provides sufficient information on internuclear distances to calculate three-dimensional structures. Figure 12.3 includes a panel showing the COSY spectrum of cyclosporin and highlights the relationships between ID H-NMR spectra and corresponding 2D homonuclear (COSY) and heteronuclear (HSQC) spectra. 

When through-bond connectivity experiments are combined with the spatial information from buildup rates of NOESY cross peaks, proton-proton distances can be obtained by comparison with known bond lengths. The result can be a complete three-dimensional structure of biomolecules. Such solution-phase structures complement solid-phase information from X-ray crystallography. In this way, NMR spectroscopy has become a structural tool for obtaining detailed molecular geometries of complex molecules in solution. 

Based on the sequence-specific assignments, two-dimensional [1H,1H]-N0E spectroscopy (NOESY) (Anil-Kumar a al, 1980) and three-dimensional N- and C-resolved NOESY (Eesik and Zuiderweg,... 

Over recent years, a range of techniques including NMR spectroscopy [45, 46] and isothermal titration calorimetry (ITC) [47,48] have been shown to provide excellent methods for the determination of binding constants for these systems. In addition, more advanced two-dimensional NMR techniques (e.g. NOESY) have been employed to ascertain the three-dimensional structure of the multicompmient complexes in solution [49], and generally show good agreement with data obtained in the solid state [50]. 

Examples of solid-state, two-dimensional NMR spectroscopy in the field of coordination chemistry are still relatively rare. The incorporation of the cross-polaiization scheme into the preparation period of the INADEQUATE,SECSY, and NOESY sequences leads to three different types of chemical-shift correlation methods, based on doublequantum, single-quantum, and spin-diffusion interactions, respectively. The former three methods rely on the presence of scalar spin-spin coupling constants of suitable size, a restriction not encountered in the spin-diffusion experiment. Overlapping resonances may be resolved with two-dimensional methods, e.g., the Pd satellites in NMR spectra of palladium phosphine or phosphite complexes see Figure 11. 

In this paper, we report the hybrid-hybrid relaxation matrix refinement tested by simulated refinement calculations on a dodecamer DNA duplex, and the refinement of the three-dimensional structure of a DNA three-way junction (TWJ), using experimental 3D NOESY-NOESY data. The TWJ has previously been studied by 2D NMR spectroscopy (12, 13). 

Overlapping resonances in 2D NMR have limited protein-structure elucidation to fairly small proteins. However, three- and four-dimensional methods have been developed that enable NMR spectroscopy to be further extended to larger and larger protein structures. A third dimension can be added, for example, to spread apart a H- H two-dimensional spectrum on the basis of the chemical shift of another nucleus, such as N or - C. In most three-dimensional experiments, the most effective methods for large molecules are used. Thus, COSY is not often employed, but experiments like NOESY-TOeSY and TOCSY-HMQC are quite effective. In some cases, the three dimensions all represent different nuclei such as H- C- N. These are considered variants of the HETCOR experiment. Multidimensional NMR is now capable of providing complete solution-phase structures to complement crystal structures from X-ray crystallography. Hence. NMR spectroscopy is now an important technique for determining structure.s and orientation.s of complex molecules in solution. 

Two-dimensional NOESY allows detection of spatial proximity between protons that are separated by less than 4.5 A. By the correlated spectroscopy method (COSY) scalar couplings between protons, which are separated by at most three (in some exceptions four) chemical bonds, are used. In the case of ribooligonucleotides such couplings consequently can only be observed between the protons of the ribose rings and the C5 and C6 protons of the pyrimidine bases of a given nucleotide. The NOESY method, however, permits the detection of contacts between the protons of different nucleotides. 

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