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nmrlearner 01-12-2015 11:31 PM

Easy and unambiguous sequential assignments of intrinsically disordered proteins by correlating the backbone 15 N or 13 Cā?² chemical shifts of multiple contiguous residues in highly resolved 3D spectra
 
Easy and unambiguous sequential assignments of intrinsically disordered proteins by correlating the backbone 15 N or 13 Cā?² chemical shifts of multiple contiguous residues in highly resolved 3D spectra

Abstract

Sequential resonance assignment strategies are typically based on matching one or two chemical shifts of adjacent residues. However, resonance overlap often leads to ambiguity in resonance assignments in particular for intrinsically disordered proteins. We investigated the potential of establishing connectivity through the three-bond couplings between sequentially adjoining backbone carbonyl carbon nuclei, combined with semi-constant time chemical shift evolution, for resonance assignments of small folded and larger unfolded proteins. Extended sequential connectivity strongly lifts chemical shift degeneracy of the backbone nuclei in disordered proteins. We show here that 3D (H)N(COCO)NH and (HN)CO(CO)NH experiments with relaxation-optimized multiple pulse mixing correlate up to seven adjacent backbone amide nitrogen or carbonyl carbon nuclei, respectively, and connections across proline residues are also obtained straightforwardly. Multiple, recurrent long-range correlations with ultra-high resolution allow backbone 1HN, 15NH, and 13Cā?² resonance assignments to be completed from a single pair of 3D experiments.



Source: Journal of Biomolecular NMR


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