Easy and unambiguous sequential assignments of intrinsically disordered proteins by correlating the backbone 15N or 13C′ chemical shifts of multiple contiguous residues in highly resolved 3D spectra
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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 13C′ resonance assignments to be completed from a single pair of 3D experiments.
KeywordsCarbonyl–carbonyl J-coupling Chemical shift degeneracy Intrinsically disordered proteins Homonuclear isotropic mixing Sequential resonance assignment
Three-bond coupling between sequentially adjoining 13C′ nuclei
Chemical shift anisotropy
Intrinsically disordered protein
Modified phase cycled Carr–Purcell multiple pulse sequence with XY16 supercycles
We thank Camilla B. Andersen and Tania A. Nielsen (Aarhus University, Denmark) for the expression and purification of αSyn and ubiquitin, respectively. Y.Y. is supported by an EMBO Long-Term Fellowship (ALTF 687-2013).
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