The complete assignment of the resonances of a protein is key to the determination of its solution structure by NMR spectroscopy and for the study of protein-protein and protein-ligand interactions. The proton-based assignment strategy usually starts with the correlation of individual resonances of each amino acid residue through scalar connectivities followed by linking them one after the other. [1,2] Although many different triple-resonance NMR spectroscopy experiments have been designed for full assignments, [2] spectral overlap can still lead to ambiguities. This poses a significant limiting factor in the cases of large and/or paramagnetic biomolecules. [3] After the pioneering report of 13 C NMR spin-system assignments of 13 C-enriched Anabaena 7120 ferredoxin by Markley and co-workers, [4] heteronuclear NMR spectroscopy experiments were progressively abandoned in favor of 1 Hdetection experiments. However, as was recently pointed out, heteronuclear NMR spectroscopy decreases the effect of detrimental transverse relaxation, which is typical of large or paramagnetic proteins. [5][6][7][8][9][10][11][12][13][14][15][16][17] For this reason, several heteronuclear NMR spectroscopy experiments for backbone assignment have been proposed for fully 13 C-and 15 N-enriched proteins. [13,14,17] Furthermore, backbone sequence-specific assignment by the recently-designed CANCO experiment has also been reported. [18] We present herein an extension of the set of exclusively heteronuclear experiments to protein side chain resonances for the complete heteronuclear assignment of a protein. With a novel CBCACO experiment the carbonyl carbon (CO) is linked to the C b and to the C a nuclei; the connection to the rest of the amino acid side chain is achieved through a 13 C-13 C TOCSY experiment with C a detection. In these experiments, we have successfully implemented spin-state selection methods for the removal of signal splitting in the acquisition dimension which is caused by multiple 13 C-13 C scalar couplings. This makes 13 C detection an amenable tool for high-resolution NMR spectroscopy. The proposed assignment strategy is summarized in Figure 1. A Figure 1. Illustration of the assignment procedure for 13 C NMR spectroscopy experiments. The assignment starts with analysis of the CACO experiment, which provides the correlation between the carbonyl carbon (CO) and the C a nuclei of each amino acid. The spin-system assignment is extended to the C b nuclei with the CBCACO experiment, and the process is completed with the TOCSY experiment, which provides correlation between the C a and the other carbon nuclei of the amino acid side chain. The amino acid spin systems are finally assigned in a sequence-specific manner with the aid of a CANCO experiment, [18] which provides the correlation of each CO to the two neighboring C a nuclei.

In magic-angle-spinning solid-state NMR, the homonuclear J-couplings between pairs of spin-1/2 nuclei may be determined by studying the modulation of the spin echo induced by a pi-pulse, as a function of the echo duration. We present the theory of J-induced spin-echo modulation in magic-angle-spinning solids, and derive a set of modulation regimes which apply under different experimental conditions. In most cases, the dominant spin-echo modulation frequency is exactly equal to the J-coupling. Somewhat surprisingly, the chemical shift anisotropies and dipole-dipole couplings tend to stabilise--rather than abscure--the J-modulation. The theoretical conclusions are supported by numerical simulations and experimental results obtained for three representative samples containing 13C spin pairs.

A routinely used assumption when interpreting two-dimensional NMR spectra obtained with a commonly used double-quantum (DQ) magic-angle-spining (MAS) pulse sequence referred to as the refocused incredible natural abundance double-quantum transfer experiment (INADEQUATE) [A. Lesage, M. Bardet, and L. Emsley, J. Am. Chem. Soc. 121, 10987 (1999)] has been that correlation peaks are only observed for pairs of nuclei with a through-bond connectivity. The validity of this assumption is addressed here by theory, experiment, and computer simulations. If the isotropic chemical shifts of the two nuclei are different and the MAS frequency is far from rotational resonance, the theoretical description demonstrates that DQ correlation peaks are indeed indicative of a J coupling. However, if the isotropic chemical shifts are the same, it is shown that DQ peaks can appear for pairs of nuclei even in the absence of a through-bond J coupling. These peaks appear in the specific case of a pair of nuclei with a nonzero through-space dipole-dipole coupling and chemical shift anisotropy tensors having different principal magnitudes or orientations, provided that the MAS frequency is comparable to or smaller than the chemical shift anisotropies. Experimental 31P spectra recorded on a sample of TiP2O7 and computer simulations show that the magnitude of these anomalous peaks increases with increasing B0 magnetic field and that they decrease with increasing MAS frequency. This behavior is explained theoretically.