After a brief theoretical description, new gradient-selected, proton-detected heteronuclear correlation sequences are introduced. The gs-HMBC and gs-Relayed-HMQC are closely related to the original gs-HMQC proposed by Hurd and John. A new approach to obtain pure absorption line shapes in gradient selected spectroscopy is used to measure phase-sensitive gs-HMQC spectra, to carry out multiplicity editing in HSQC spectra and to distinguish direct and long-range correlations in HMQC/HSQC-TOCSY spectra.

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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.

Natively unfolded proteins are increasingly recognized to play important physiological roles. These proteins do not crystallize, so NMR is the only technique able to provide structural and dynamic information. However, in unfolded proteins, the proton chemical shift dispersion is poor, causing severe problems in resonance assignment. We designed a novel strategy based on two protonless experiments, a CBCACON-IPAP and a novel COCON-IPAP, that permits a straightforward and unequivocal backbone heteronuclear assignment of the natively unfolded protein alpha-synuclein.