13C-detected protonless NMR spectroscopy of proteins in solution

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Why the need of hetero detection

The NMR determination of the structure of large biological macromolecules in solution is primarily limited by the fast transverse relaxation that broadens lines and reduces spectral resolution. Several steps ahead have been made recently to overcome this limitation. In particular, the constructive use of cross-correlated relaxation phenomena enables a reduction of the effective transverse relaxation rates of specific spins, such as backbone NH groups [1] and aromatic CH groups [2]. More

Instrumental aspects

The sensitivity in NMR experiments is given by Eq. (1)S/NAγexcγobs3/2B03/2Nscan1/2were γexc and γobs are the gyromagnetic ratios of the excited and of the observed nuclei, respectively, A is the number of spins, Nscan is the number of scans and B0 is the strength of the applied magnetic field [23]. Therefore, neglecting relaxation, 1H observed experiments have a sensitivity gain of a factor of 32 (i.e. (γH/γC)5/2) compared to the corresponding experiments starting and ending at 13C nuclei

The problem of the 13C–13C coupling

In 13C direct-detection experiments we need to face the problem of the large homonuclear one-bond carbon–carbon couplings that evolve during the acquisition delay. The presence of these couplings is, of course, beneficial for coherence transfer efficiency but is detrimental to resolution in the acquisition dimension. In addition to the large one-bond 13C homonuclear scalar couplings (1JCC=35–55 Hz), there are also a variety of smaller homonuclear scalar couplings, such as 2JCC, 3JCC, and 4JCC,

A protocol for the assignment of backbone and side chains

The methods described above to detect selected 13C nuclear spins whilst collapsing the large one-bond homonuclear carbon–carbon scalar couplings, as well as the availability of probeheads with improved sensitivity for 13C [14], [53], have opened the way for the use of 13C direct-detection experiments in biomolecular NMR. We summarize here a set of experiments based exclusively on heteronuclei that allows one to perform a complete sequence specific assignment of a 13C,15N labeled protein without

Detection of resonances in paramagnetic proteins

Paramagnetic systems are characterized by additional contributions to chemical shifts and nuclear relaxation arising from the so-called hyperfine interaction, i.e. the interaction between the nuclear spin I and the electron spin S [68]. This interaction occurs via two different mechanisms, a through-bond interaction, which depends on the amount of unpaired electron spin-density delocalized onto the investigated nuclear spin (contact interaction) [69], [70] and a through-space interaction [71],

Conclusions

We have shown that the combination of upgraded instrumental performances and the design of novel pulse sequences allows 13C direct-detected protonless NMR spectroscopy to be used to conveniently perform the complete 13C,15N assignment for proteins. Development of the exclusively heteronuclear NMR experiments summarized in this review has been stimulated by the need for a different approach to study paramagnetic systems, for which the main problems are fast transverse relaxation and difficulties

Acknowledgements

This work has been supported in part by the EC (Contract QLG2-CT-2002-00988) and by the Italian Ministero per la Università e la Ricerca (COFIN 2003).

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