Journal of Biomolecular NMR

, Volume 61, Issue 2, pp 109–121 | Cite as

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

  • Yuichi Yoshimura
  • Natalia V. Kulminskaya
  • Frans A. A. Mulder


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.


Carbonyl–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).

Supplementary material

10858_2014_9890_MOESM1_ESM.docx (34 kb)
Supplementary material 1 (DOCX 34 kb)


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Copyright information

© Springer Science+Business Media Dordrecht 2015

Authors and Affiliations

  • Yuichi Yoshimura
    • 1
  • Natalia V. Kulminskaya
    • 1
  • Frans A. A. Mulder
    • 1
  1. 1.Department of Chemistry and Interdisciplinary Nanoscience Center (iNANO)Aarhus UniversityAarhus CDenmark

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