Journal of Biomolecular NMR

, Volume 50, Issue 1, pp 1–11 | Cite as

5D 13C-detected experiments for backbone assignment of unstructured proteins with a very low signal dispersion

  • Jiří Nováček
  • Anna Zawadzka-Kazimierczuk
  • Veronika Papoušková
  • Lukáš Žídek
  • Hana Šanderová
  • Libor Krásný
  • Wiktor Koźmiński
  • Vladimír Sklenář


Two novel 5D NMR experiments (CACONCACO, NCOCANCO) for backbone assignment of disordered proteins are presented. The pulse sequences exploit relaxation properties of the unstructured proteins and combine the advantages of 13C-direct detection, non-uniform sampling, and longitudinal relaxation optimization to maximize the achievable resolution and minimize the experimental time. The pulse sequences were successfully tested on the sample of partially disordered delta subunit from RNA polymerase from Bacillus subtilis. The unstructured part of this 20 kDa protein consists of 81 amino acids with frequent sequential repeats. A collection of 0.0003% of the data needed for a conventional experiment with linear sampling was sufficient to perform an unambiguous assignment of the disordered part of the protein from a single 5D spectrum.


Intrinsically disordered proteins Non-uniform sampling 13C detection Longitudinal relaxation optimization Backbone assignment 



This work was supported by the Grants of the Ministry of Education of Czech Republic MSM0021622413 and LC06030, by the Grants 204/09/0583, 301/09/H004 and P206/11/0758 from Czech Science Foundation, by the EU/ grant POSTBIOMIN (FP7-REGPOT-2007-1 No. 205872), by MPD program from Foundation for Polish Sciences that was co-financed by the European Regional Development Fund. Financial support including the form of Access to the Bio-NMR Research Infrastructure co-funded under the 7th Framework Programme of the EC (FP7/2007-2013) grant agreement 261863 for conducting the research is gratefully acknowledged.

Supplementary material

10858_2011_9496_MOESM1_ESM.pdf (313 kb)
PDF (312 KB)


  1. Achberger EC, Hilton MD, Whiteley HR (1982) The effect of the delta subunit on the interaction of Bacillus subtilis RNA polymerase with bases in a SP82 early gene promoter. Nucl Acids Res 10:2893–2910CrossRefGoogle Scholar
  2. Atreya HS, Szyperski T (2004) G-matrix Fourier transform NMR spectroscopy for complete protein resonance assignment. PNAS 101(26):9642–9647ADSCrossRefGoogle Scholar
  3. Atreya H, Eletsky A, Szyperski T (2005) Resonance assignment of proteins with high shift degeneracy based on 5D spectral information encoded in G(2)FT NMR experiments. J Am Chem Soc 127(13):4554–4555CrossRefGoogle Scholar
  4. Bermel W, Bertini I, Duma L, Felli IC, Emsley L, Pierattelli R, Vasos PR (2005) Complete assignment of heteronuclear protein resonances by protonless NMR spectroscopy. Angew Chem Int Ed 44(20):3089–3092CrossRefGoogle Scholar
  5. Bermel W, Bertini I, Felli IC, Piccioli M, Pierattelli R (2006) C-13-detected protonless NMR spectroscopy of proteins in solution. Prog Nucl Magn Reson Spectrosc 48(1):25–45CrossRefGoogle Scholar
  6. Bermel W, Bertini I, Felli I, Lee Y, Luchinat C, Pierattelli R (2006) Protonless NMR experiments for sequence-specific assignment of backbone nuclei in unfolded proteins. J Am Chem Soc 128(12):3918–3919CrossRefGoogle Scholar
  7. Bermel W, Bertini I, Felli IC, Piccioli M, Pierattelli R (2009) Speeding up C-13 direct detection biomolecular NMR spectroscopy. J Am Chem Soc 131(42):15339–15345CrossRefGoogle Scholar
  8. Bretthorst GL (2008) Nonuniform sampling: bandwidth and aliasing. Concepts Magn Reson 32A(6):417–435CrossRefGoogle Scholar
  9. Brutscher B (2002) Intraresidue HNCA and COHNCA experiments for protein backbone resonance assignment. J Magn Reson 156(1):155–159ADSCrossRefGoogle Scholar
  10. Delaglio F, Grzesiek S, Vuister G, Zhu G, Pfeifer J, Bax A (1995) NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR 6(3):277–293CrossRefGoogle Scholar
  11. Dunker AK, Obradovic Z, Romero P, Garner EC, Brown CJ (2000) Intrinsic protein disorder in complete genomes. Genome Inform 11:161–171Google Scholar
  12. Eliezer D (2007) Characterizing residual structure in disordered protein states using nuclear magnetic resonance. Methods Mol Biol 350:49–67Google Scholar
  13. Fiorito F, Hiller S, Wider G, Wüthrich K (2006) Automated resonance assignment of proteins: 6D APSY-NMR. J Biomol NMR 35(1):27–37CrossRefGoogle Scholar
  14. Freeman R, Kupče E (2004) Distant echoes of the accordion: reduced dimensionality, GFT-NMR, and projection-reconstruction of multidimensional spectra. Concepts Mang Reson 23(2):63–75CrossRefGoogle Scholar
  15. Frueh DP, Sun ZYJ, Vosburg DA, Walsh CT, Hoch JC, Wagner G (2006) Non-uniformly sampled double-TROSY hNcaNH experiments for NMR sequential assignments of large proteins. J Am Chem Soc 128(17):5757–5763CrossRefGoogle Scholar
  16. Hiller S, Fiorito F, Wuthrich K, Wider G (2005) Automated projection spectroscopy (APSY). PNAS 102(31):10876–10881ADSCrossRefGoogle Scholar
  17. Hiller S, Wasmer C, Wider G, Wüthrich K (2007) Sequence-specific resonance assignment of soluble nonglobular proteins by 7D APSY-NMR spectroscopy. J Am Chem Soc 129(35):10823–10828CrossRefGoogle Scholar
  18. Kazimierczuk K, Zawadzka A, Koźmiński W, Zhukov I (2007) Lineshapes and artifacts in Multidimensional Fourier Transform of arbitrary sampled NMR data sets. J Magn Reson 188(2):344–356ADSCrossRefGoogle Scholar
  19. Kazimierczuk K, Zawadzka A, Koźmiński W (2008) Optimization of random time domain sampling in multidimensional NMR. J Magn Reson 192(1):123–130ADSCrossRefGoogle Scholar
  20. Kazimierczuk K, Zawadzka A, Koźmiński W (2009) Narrow peaks and high dimensionalities: exploiting the advantages of random sampling. J Magn Reson 205(2):286–292ADSCrossRefGoogle Scholar
  21. Kazimierczuk K, Zawadzka-Kazimierczuk A, Koźmiński W (2010) Non-uniform frequency domain for optimal exploitation of non-uniform sampling. J Magn Reson 197(2):219–228ADSCrossRefGoogle Scholar
  22. Knoblich K, Whittaker S, Ludwig C, Michiels P, Jiang T, Schaffhausen B, Guenther U (2009) Backbone assignment of the N-terminal polyomavirus large T antigen. Biomol NMR Assign 3(1):119–123CrossRefGoogle Scholar
  23. Malmodin D, Billeter M (2005) Multiway decomposition of NMR spectra with coupled evolution periods. J Am Chem Soc 127(39):13486–13487CrossRefGoogle Scholar
  24. Marion D (2006) Processing of ND NMR spectra sampled in polar coordinates: a simple Fourier transform instead of a reconstruction. J Biomol NMR 36(1):45–54CrossRefGoogle Scholar
  25. Mobli M, Hoch JC (2008) Maximum entropy spectral reconstruction of nonuniformly sampled data. Concepts Magn Reson 32A(6):436–448CrossRefGoogle Scholar
  26. Motáčková V, Kubíčková M, Kožíšek M, Grantz-Šašková K, Švec M, Žídek L, Sklenář V (2009) Backbone H-1, C-13, and N-15 NMR assignment for the inactive form of the retroviral protease of the murine intracisternal A-type particle, inMIA-14 PR. Biomol NMR Assign 3(2):261–264CrossRefGoogle Scholar
  27. Motáčková V, Šanderová H, Žídek L, Nováček J, Padrta P, Švenková A, Korelusová J, Jonák J, Krásný L, Sklenář V (2010) Solution structure of the N-terminal domain of Bacillus subtilis delta subunit of RNA polymerase and its classification based on structural homologs. Proteins Struct Funct Bioinf 78(7):1807–1810Google Scholar
  28. Motáčková V, Nováček J, Zawadzka-Kazimierczuk A, Kazimierczuk K, Žídek L, Koźmiński W, Sklenář V (2010) Strategy for complete NMR assignment of disordered proteins with highly repetitive sequences based on resolution-enhanced 5D experiments. J Biomol NMR 48(3):169–177CrossRefGoogle Scholar
  29. Mukrasch M, Bibow S, Korukottu J, Jeganathan S, Biernat J, Griesinger C, Mandelkow E, Zweckstetter M (2009) Structural polymorphism of 441-residue Tau at single sesidue resolution. PLoS Biol 7(2):399–414CrossRefGoogle Scholar
  30. Narayanan RL, Durr UHN, Bibow S, Biernat J, Mandelkow E3, Zweckstetter M (2010) Automatic assignment of the intrinsically disordered protein tau with 441-residues. J Am Chem Soc 132(34):11906–11907CrossRefGoogle Scholar
  31. Orekhov VY, Ibraghimov IV, Billeter M (2001) MUNIN: a new approach to multi-dimensional NMR spectra interpretation. J Biomol NMR 20(1):49–60CrossRefGoogle Scholar
  32. Panchal SC, Bhavesh NS, Hosur RV (2001) Improved 3D triple resonance experiments, HNN and HN(C)N, for H-N and N-15 sequential correlations in (C-13, N-15) labeled proteins: application to unfolded proteins. J Biomol NMR 20(2):135–147CrossRefGoogle Scholar
  33. Pannetier N, Houben K, Blanchard L, Marion D (2007) Optimized 3D-NMR sampling for resonance assignment of partially unfolded proteins. J Magn Reson 186(1):142–149ADSCrossRefGoogle Scholar
  34. Perez Y, Gairi M, Pons M, Bernado P (2009) Structural characterization of the natively unfolded N-terminal domain of human c-Src kinase: insights into the role of phosphorylation of the unique domain. J Mol Biol 391(1):136–148CrossRefGoogle Scholar
  35. Pervushin K, Vogeli B, Eletsky A (2002) Longitudinal H-1 relaxation optimization in TROSY NMR spectroscopy. J Am Chem Soc 124(43):12898–12902CrossRefGoogle Scholar
  36. Peti W, Smith LJ, Redfield C, Schwalbe H (2001) Chemical shifts in denatured proteins: resonance assignments for denatured ubiquitin and comparisons with other denatured proteins. J Biomol NMR 19(2):153–165CrossRefGoogle Scholar
  37. Rovnyak D, Frueh DP, Sastry M, Sun ZYJ, Stern AS, Hoch JC, Wagner G (2004) Accelerated acquisition of high resolution triple-resonance spectra using non-uniform sampling and maximum entropy reconstruction. J Magn Reson 170(1):15–21ADSCrossRefGoogle Scholar
  38. Sattler M, Schleucher J, Griesinger C (1999) Heteronuclear multidimensional NMR experiments for the structure determination of proteins in solution employing pulsed field gradients. Prog Nucl Mang Reson Spect 34(2):93–158CrossRefGoogle Scholar
  39. Schanda P, Brutscher B (2005) Very fast two-dimensional NMR spectroscopy for real-time investigation of dynamic events in proteins on the time scale of seconds. J Am Chem Soc 127(22):8014–8015CrossRefGoogle Scholar
  40. Seepersaud R, Needham RHV, Kim CS, Jones AL (2006) Abundance of the δ subunit of RNA polymerase is linked to the virulence of Streptococcus agalactiae. J Bacteriol 188(8):2096–2105CrossRefGoogle Scholar
  41. Sklenář V (1995) Suppression of radiation damping in multidimensional NMR experiments using magentic-field gradients. J Magn Reson Ser A 114(1):132–135CrossRefGoogle Scholar
  42. Sørensen OW, Eich GW, Levitt MH, Bodenhausen G, Ernst RR (1984) Product operator formalism for the description of NMR pulse experiments. Prog Nucl Mang Reson Spect 16:163–192CrossRefGoogle Scholar
  43. Stern AS, Li KB, Hoch JC (2002) Modern spectrum analysis in multidimensional NMR spectroscopy: comparison of linear-prediction extrapolation and maximum-entropy reconstruction. J Am Chem Soc 124(9):1982–1993CrossRefGoogle Scholar
  44. Sun ZYJ, Frueh DP, Selenko P, Hoch JC, Wagner G (2005) Fast assignment of N-15-HSQC peaks using high-resolution 3D HNcocaNH experiments with non-uniform sampling. J Biomol NMR 33(1):43–50CrossRefGoogle Scholar
  45. Ward JJ, Sodhi JS, McGuffin LJ, Buxton BF, Jones DT (2004) Prediction and functional analysis of native disorder in proteins from the three kingdoms of life. J Mol Biol 337(3):635–645CrossRefGoogle Scholar
  46. Yao J, Chung J, Eliezer D, Wright PE, Dyson HJ (2001) NMR structural and dynamic characterization of the acid-unfolded state of apomyoglobin provides insights into the early events in protein folding. Biochemistry 40(12):3561–3571CrossRefGoogle Scholar
  47. Zawadzka-Kazimierczuk A, Kazimierczuk K, Koźmiński W (2010) A set of 4D NMR experiments of enhanced resolution for easy resonance assignment in proteins. J Magn Reson 202(1):109–116ADSCrossRefGoogle Scholar
  48. Zweckstetter M, Bax A (2001) Single-step determination of protein substructures using dipolar couplings: aid to structural genomics. J Am Chem Soc 123(39):9490–9491CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Jiří Nováček
    • 1
  • Anna Zawadzka-Kazimierczuk
    • 2
  • Veronika Papoušková
    • 1
  • Lukáš Žídek
    • 1
  • Hana Šanderová
    • 3
  • Libor Krásný
    • 3
  • Wiktor Koźmiński
    • 2
  • Vladimír Sklenář
    • 1
  1. 1.Faculty of Science, NCBR, and CEITECMasaryk UniversityBrnoCzech Republic
  2. 2.Faculty of ChemistryUniversity of WarsawWarsawPoland
  3. 3.Laboratory of Molecular Genetics of Bacteria and Department of BacteriologyInstitute of Microbiology, Academy of Sciences of the Czech RepublicPragueCzech Republic

Personalised recommendations