Journal of Biomolecular NMR

, Volume 63, Issue 2, pp 201–212 | Cite as

HN-NCA heteronuclear TOCSY-NH experiment for 1HN and 15N sequential correlations in (13C, 15N) labelled intrinsically disordered proteins

  • Christoph Wiedemann
  • Nishit Goradia
  • Sabine Häfner
  • Christian Herbst
  • Matthias Görlach
  • Oliver Ohlenschläger
  • Ramadurai RamachandranEmail author


A simple triple resonance NMR experiment that leads to the correlation of the backbone amide resonances of each amino acid residue ‘i’ with that of residues ‘i−1’ and ‘i+1’ in (13C, 15N) labelled intrinsically disordered proteins (IDPs) is presented. The experimental scheme, {HN-NCA heteronuclear TOCSY-NH}, exploits the favourable relaxation properties of IDPs and the presence of 1 J CαN and 2 J CαN couplings to transfer the 15N x magnetisation from amino acid residue ‘i’ to adjacent residues via the application of a band-selective 15N–13Cα heteronuclear cross-polarisation sequence of ~100 ms duration. Employing non-uniform sampling in the indirect dimensions, the efficacy of the approach has been demonstrated by the acquisition of 3D HNN chemical shift correlation spectra of α-synuclein. The experimental performance of the RF pulse sequence has been compared with that of the conventional INEPT-based HN(CA)NH pulse scheme. As the availability of data from both the HCCNH and HNN experiments will make it possible to use the information extracted from one experiment to simplify the analysis of the data of the other and lead to a robust approach for unambiguous backbone and side-chain resonance assignments, a time-saving strategy for the simultaneous collection of HCCNH and HNN data is also described.


NMR spectroscopy Intrinsically disordered protein region Heteronuclear cross polarisation Sequential resonance assignment Non-uniform sampling 



The FLI is a member of the Science Association ‘Gottfried Wilhelm Leibniz’ (WGL) and is financially supported by the Federal Government of Germany and the State of Thuringia. We are deeply grateful to Dr. Lisa D. Cabrita and Prof. Dr. John Christodoulou (Insitute of Structural and Molecular Biology, University College London and Birkbeck College, London, United Kingdom) for providing us with the plasmid and purification protocol for the α-synuclein sample used in this study. Financial support by the Deutsche Forschungsgemeinschaft (DFG) within FOR 1738 (to O.O. and N.G.) is gratefully acknowledged.

Supplementary material

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Supplementary material 1 (DOCX 2081 kb)


  1. Bellstedt P, Ihle Y, Wiedemann C, Kirschstein A, Herbst C, Görlach M, Ramachandran R (2014) Sequential acquisition of multi-dimensional heteronuclear chemical shift correlation spectra with 1H detection. Sci Rep 4:4490CrossRefADSGoogle Scholar
  2. Bermel W, Bertini I, Felli IC, Lee YM, Luchinat C, Pierattelli R (2006) Protonless NMR experiments for sequence-specific assignment of backbone nuclei in unfolded proteins. J Am Chem Soc 128:3918–3919CrossRefGoogle Scholar
  3. Bermel W, Bruix M, Felli IC, Kumar MVV, Pierattelli R, Serrano S (2013a) Improving the chemical shift dispersion of multidimensional NMR spectra of intrinsically disordered proteins. J Biomol NMR 55:231–237CrossRefGoogle Scholar
  4. Bermel W, Felli IC, Gonnelli L, Koźmiński W, Piai A, Pierattelli R, Zawadzka-Kazimierczuk A (2013b) High-dimensionality 13C direct-detected NMR experiments for the automatic assignment of intrinsically disordered proteins. J Biomol NMR 57:353–361CrossRefGoogle Scholar
  5. Bracken C, Palmer AG 3rd, Cavanagh J (1997) (H)N(COCA)NH and HN(COCA)NH experiments for 1H–15N backbone assignments in 13C/15N-labeled proteins. J Biomol NMR 9:94–100CrossRefGoogle Scholar
  6. Chandra K, Jaipuria G, Shet D, Atreya HS (2012) Efficient sequential assignments in proteins with reduced dimensionality 3D HN(CA)NH. J Biomol NMR 52:115–126CrossRefGoogle Scholar
  7. Clowes RT, Boucher W, Hardman CH, Domaille PJ, Laue ED (1993) A 4D HCC(CO)NNH experiment for the correlation of aliphatic side-chain and backbone resonances in 13C/15N-labelled proteins. J Biomol NMR 3:349–354Google Scholar
  8. Felli IC, Pierattelli R (2014) Novel methods based on 13C detection to study intrinsically disordered proteins. J Magn Reson 241:115–125CrossRefADSGoogle Scholar
  9. Felli IC, Pierattelli R, Tompa P (2012) Intrinsically Disordered Proteins. In: Bertini I, McGreevy KS, Parigi G (eds) NMR of biomolecules: towards mechanistic systems biology, First Edition edn. Wiley-VCH Verlag & Co. KGaA, Weinheim, pp 137–152Google Scholar
  10. Gil S, Hošek T, Solyom Z, Kümmerle R, Brutscher B, Pierattelli R, Felli IC (2013) NMR spectroscopic studies of intrinsically disordered proteins at near-physiological conditions. Angew Chem Int Ed Engl 52:11808–11812CrossRefGoogle Scholar
  11. Glaser SJ, Quant JJ (1996) Homonuclear and heteronuclear Hartmann–Hahn transfer in isotropic liquids. Adv Magn Reson 19:59–252CrossRefGoogle Scholar
  12. Goradia N, Wiedemann C, Herbst C, Görlach M, Heinemann SH, Ohlenschläger O, Ramachandran R (2015) An approach to NMR assignment of intrinsically disordered proteins. ChemPhysChem 16:739–746CrossRefGoogle Scholar
  13. Grzesiek S, Anglister J, Bax A (1993) Correlation of backbone amide and aliphatic side-chain resonances in 13C/15N-enriched proteins by isotropic mixing of 13C magnetization. J Magn Reson B 101:114–119CrossRefGoogle Scholar
  14. Hellman M, Tossavainen H, Rappu P, Heino J, Permi P (2011) Characterization of intrinsically disordered prostate associated gene (PAGE5) at single residue resolution by NMR spectroscopy. PLoS One 6:e26633CrossRefADSGoogle Scholar
  15. Hellman M, Piirainen H, Jaakola VP, Permi P (2014) Bridge over troubled proline: assignment of intrinsically disordered proteins using (HCA)CON(CAN)H and (HCA)N(CA)CO(N)H experiments concomitantly with HNCO and i(HCA)CO(CA)NH. J Biomol NMR 58:49–60CrossRefGoogle Scholar
  16. Hoyer W, Antony T, Cherny D, Heim G, Jovin TM, Subramaniam V (2002) Dependence of alpha-synuclein aggregate morphology on solution conditions. J Mol Biol 322:383–393CrossRefGoogle Scholar
  17. Hsu ST, Bertoncini CW, Dobson CM (2009) Use of protonless NMR spectroscopy to alleviate the loss of information resulting from exchange-broadening. J Am Chem Soc 131:7222–7223CrossRefGoogle Scholar
  18. Hyberts SG, Arthanari H, Robson SA, Wagner G (2014) Perspectives in magnetic resonance: NMR in the post-FFT era. J Magn Reson 241:60–73CrossRefADSGoogle Scholar
  19. Ikegami T, Sato S, Wälchli M, Kyogoku Y, Shirakawa M (1997) An efficient HN(CA)NH pluse scheme for triple-resonance 4D correlation of sequential amide protons and nitrogens-15 in deuterated proteins. J Magn Reson 124:214–217CrossRefADSGoogle Scholar
  20. Isaksson L, Mayzel M, Saline M, Pedersen A, Rosenlöw J, Brutscher B, Karlsson BG, Orekhov VY (2013) Highly efficient NMR assignment of intrinsically disordered proteins: application to B- and T cell receptor domains. PLoS One 8:e62947CrossRefADSGoogle Scholar
  21. Jaravine V, Ibraghimov I, Orekhov VY (2006) Removal of a time barrier for high-resolution multi-dimensional NMR spectroscopy. Nat Methods 3:605–607CrossRefGoogle Scholar
  22. Jensen MR, Ruigrok RW, Blackledge M (2013) Describing intrinsically disordered proteins at atomic resolution by NMR. Curr Opin Struct Biol 23:426–435CrossRefGoogle Scholar
  23. Kazimierczuk K, Orekhov VY (2011) Accelerated NMR spectroscopy by using compressed sensing. Angew Chem Int Ed Engl 50:5556–5559CrossRefGoogle Scholar
  24. Kirschstein A, Herbst C, Riedel K, Carella M, Leppert J, Ohlenschläger O, Görlach M, Ramachandran R (2008a) Broadband homonuclear TOCSY with amplitude and phase-modulated RF mixing schemes. J Biomol NMR 40:227–237CrossRefGoogle Scholar
  25. Kirschstein A, Herbst C, Riedel K, Carella M, Leppert J, Ohlenschläger O, Görlach M, Ramachandran R (2008b) Heteronuclear J cross-polarisation in liquids using amplitude and phase modulated mixing sequences. J Biomol NMR 40:277–283CrossRefGoogle Scholar
  26. Konrat R (2014) NMR contributions to structural dynamics studies of intrinsically disordered proteins. J Magn Reson 241:74–85CrossRefADSGoogle Scholar
  27. Kosol S, Contreras-Martos S, Cedeño C, Tompa P (2013) Structural characterization of intrinsically disordered proteins by NMR spectroscopy. Molecules 18:10802–10828CrossRefGoogle Scholar
  28. Liu X, Yang D (2013) HN(CA)N and HN(COCA)N experiments for assignment of large disordered proteins. J Biomol NMR 57:83–89CrossRefGoogle Scholar
  29. Logan TM, Olejniczak ET, Xu RX, Fesik SW (1992) Side chain and backbone assignments in isotropically labeled proteins from two heteronuclear triple resonance experiments. FEBS Lett 314:413–418CrossRefGoogle Scholar
  30. Luan T, Jaravine V, Yee A, Arrowsmith CH, Orekhov VY (2005) Optimization of resolution and sensitivity of 4D NOESY using multidimensional decomposition. J Biomol NMR 33:1–14CrossRefGoogle Scholar
  31. Lyons BA, Montelione GT (1993) An HCCNH triple-resonance experiment using carbon-13 isotropic mixing for correlating backbone amide and side-chain aliphatic resonances in isotopically enriched proteins. J Magn Reson B 101:206–209CrossRefGoogle Scholar
  32. Maciejewski MW, Mobli M, Schuyler AD, Stern AS, Hoch JC (2012) Data sampling in multidimensional NMR: fundamentals and strategies. Top Curr Chem 316:49–77CrossRefGoogle Scholar
  33. Mäntylahti S, Aitio O, Hellman M, Permi P (2010) HA-detected experiments for the backbone assignment of intrinsically disordered proteins. J Biomol NMR 47:171–181CrossRefGoogle Scholar
  34. Mäntylahti S, Hellman M, Permi P (2011) Extension of the HA-detection based approach: (HCA)CON(CA)H and (HCA)NCO(CA)H experiments for the main-chain assignment of intrinsically disordered proteins. J Biomol NMR 49:99–109CrossRefGoogle Scholar
  35. Motáčková V, Nováček J, Zawadzka-Kazimierczuk A, Kazimierczuk K, Žídek L, Šanderová H, Krásný 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 48:169–177CrossRefGoogle Scholar
  36. Narayanan RL, Dürr UH, Bibow S, Biernat J, Mandelkow E, Zweckstetter M (2010) Automatic assignment of the intrinsically disordered protein Tau with 441-residues. J Am Chem Soc 132:11906–11907CrossRefGoogle Scholar
  37. Nováček J, Haba NY, Chill JH, Židek L, Sklenář V (2012) 4D non-uniformly sampled HCBCACON and 1JNCα-selective HCBCANCO experiments for the sequential assignment and chemical shift analysis of intrinsically disordered proteins. J Biomol NMR 53:139–148CrossRefGoogle Scholar
  38. Nováček J, Janda L, Dopitová R, Židek L, Sklenář V (2013) Efficient protocol for backbone and side-chain assignments of large, intrinsically disordered proteins: transient secondary structure analysis of 49.2 kDa microtubule associated protein 2c. J Biomol NMR 56:291–301CrossRefGoogle Scholar
  39. Nováček J, Židek L, Sklenář V (2014) Toward optimal-resolution NMR of intrinsically disordered proteins. J Magn Reson 241:41–52CrossRefADSGoogle Scholar
  40. Orekhov VY, Ibragimov I, Billeter M (2003) Optimizing resolution in multidimensional NMR by threeway decomposition. J Biomol NMR 27:165–173CrossRefGoogle Scholar
  41. Palmer MR, Wenrich BR, Stahlfeld P, Rovnyak D (2014) Performance tuning non-uniform sampling for sensitivity enhancement of signal-limited biological NMR. J Biomol NMR 58:303–314CrossRefGoogle Scholar
  42. Panchal SC, Bhavesh NS, Hosur RV (2001) Improved 3D triple resonance experiments, HNN and HN(C)N, for HN and 15N sequential correlations in (13C, 15N) labeled proteins: application to unfolded proteins. J Biomol NMR 20:135–147CrossRefGoogle Scholar
  43. Pantoja-Uceda D, Santoro J (2013) Direct correlation of consecutive C’–N groups in proteins: a method for the assignment of intrinsically disordered proteins. J Biomol NMR 57:57–63CrossRefGoogle Scholar
  44. Pantoja-Uceda D, Santoro J (2014) New 13C-detected experiments for the assignment of intrinsically disordered proteins. J Biomol NMR 59:43–50CrossRefGoogle Scholar
  45. Paramasivam S, Suiter CL, Hou G, Sun S, Palmer M, Hoch JC, Rovnyak D, Polenova T (2012) Enhanced sensitivity by nonuniform sampling enables multidimensional MAS NMR spectroscopy of protein assemblies. J Phys Chem B 116:7416–7427CrossRefGoogle Scholar
  46. Piai A, Hošek T, Gonnelli L, Zawadzka-Kazimierczuk A, Koźmiński W, Brutscher B, Bermel W, Pierattelli R, Felli IC (2014) “CON-CON” assignment strategy for highly flexible intrinsically disordered proteins. J Biomol NMR 60:209–218CrossRefGoogle Scholar
  47. Reddy JG, Hosur RV (2014) A reduced dimensionality NMR pulse sequence and an efficient protocol for unambiguous assignment in intrinsically disordered proteins. J Biomol NMR 59:199–210CrossRefGoogle Scholar
  48. Rezaei-Ghaleh N, Blackledge M, Zweckstetter M (2012) Intrinsically disordered proteins: from sequence and conformational properties toward drug discovery. ChemBioChem 13:930–950CrossRefGoogle Scholar
  49. Richardson JM, Clowes RT, Boucher W, Domaille PJ, Hardman CH, Keeler J, Laue ED (1993) The use of heteronuclear cross polarization to enhance the sensitivity of triple-resonance NMR experiments. Improved 4D HCNNH pulse sequences. J Magn Reson B 101:223–227Google Scholar
  50. Rovnyak D, Sarcone M, Jiang Z (2011) Sensitivity enhancement for maximally resolved two-dimensional NMR by nonuniform sampling. Magn Reson Chem 49:483–491CrossRefGoogle Scholar
  51. Sahoo N, Goradia N, Ohlenschläger O, Schönherr R, Friedrich M, Plass W, Kappl R, Hoshi T, Heinemann SH (2013) Heme impairs the ball-and-chain inactivation of potassium channels. Proc Natl Acad Sci USA 110:E4036–E4044CrossRefADSGoogle Scholar
  52. Sahu D, Bastidas M, Showalter SA (2014) Generating NMR chemical shift assignments of intrinsically disordered proteins using carbon-detected NMR methods. Anal Biochem 449:17–25CrossRefGoogle Scholar
  53. Salmon L, Nodet G, Ozenne V, Yin G, Jensen MR, Zweckstetter M, Blackledge M (2010) NMR characterization of long-range order in intrinsically disordered proteins. J Am Chem Soc 132:8407–8418CrossRefGoogle Scholar
  54. Schulenburg C, Hilvert D (2013) Protein conformational disorder and enzyme catalysis. Top Curr Chem 337:41–67CrossRefGoogle Scholar
  55. Shirakawa M, Wälchli M, Shimizu M, Kyogoku Y (1995) The use of heteronuclear cross-polarization for backbone assignment of 2H-, 15N- and 13C-labeled proteins: a pulse scheme for triple-resonance 4D correlation of sequential amide protons and 15N. J Biomol NMR 5:323–326CrossRefGoogle Scholar
  56. Sibille N, Bernadó P (2012) Structural characterization of intrinsically disordered proteins by the combined use of NMR and SAXS. Biochem Soc Trans 40:955–962CrossRefGoogle Scholar
  57. Skrabana R, Sevcik J, Novak M (2006) Intrinsically disordered proteins in the neurodegenerative processes: formation of tau protein paired helical filaments and their analysis. Cell Mol Neurobiol 26:1085–1097CrossRefGoogle Scholar
  58. Solyom Z, Schwarten M, Geist L, Konrat R, Willbold D, Brutscher B (2013) BEST-TROSY experiments for time-efficient sequential resonance assignment of large disordered proteins. J Biomol NMR 55:311–321CrossRefGoogle Scholar
  59. States DJ, Haberkorn RA, Ruben DJ (1982) A two-dimensional nuclear overhauser experiment with pure absorption phase in four quadrants. J Magn Reson 48:286–292ADSGoogle Scholar
  60. Szalainé Ágoston B, Kovács D, Tompa P, Perczel A (2011) Full backbone assignment and dynamics of the intrinsically disordered dehydrin ERD14. Biomol NMR Assign 5:189–193CrossRefGoogle Scholar
  61. Theillet FX, Binolfi A, Liokatis S, Verzini S, Selenko P (2011) Paramagnetic relaxation enhancement to improve sensitivity of fast NMR methods: application to intrinsically disordered proteins. J Biomol NMR 51:487–495CrossRefGoogle Scholar
  62. Tompa P (2011) Unstructural biology coming of age. Curr Opin Struct Biol 21:419–425CrossRefGoogle Scholar
  63. Tompa P (2012) Intrinsically disordered proteins: a 10-year recap. Trends Biochem Sci 37:509–516CrossRefGoogle Scholar
  64. Tugarinov V, Kay LE, Ibraghimov I, Orekhov VY (2005) High-resolution four-dimensional 1H–13C NOE spectroscopy using methyl-TROSY, sparce data acquisition, and multidimensional decomposition. J Am Chem Soc 127:2767–2775CrossRefGoogle Scholar
  65. Uversky VN, Oldfield CJ, Midic U, Xie H, Xue B, Vucetic S, Iakoucheva LM, Obradovic Z, Dunker AK (2009) Unfoldomics of human diseases: linking protein intrinsic disorder with diseases. BMC Genom 10(Suppl 1):S7CrossRefGoogle Scholar
  66. Waudby CA, Camilloni C, Fitzpatrick AW, Cabrita LD, Dobson CM, Vendruscolo M, Christodoulou J (2013) In-cell NMR characterization of the secondary structure populations of a disordered conformation of alpha-synuclein within E. coli cells. PLoS One 8:e72286CrossRefADSGoogle Scholar
  67. Weisemann R, Rüterjans H, Bermel W (1993) 3D triple-resonance NMR techniques for the sequential assignment of NH and 15N resonances in 15N- and 13C-labelled proteins. J Biomol NMR 3:113–120CrossRefGoogle Scholar
  68. Wen J, Wu J, Zhou P (2011) Sparsely sampled high-resolution 4-D experiments for efficient backbone resonance assignment of disordered proteins. J Magn Reson 209:94–100CrossRefADSGoogle Scholar
  69. Wiedemann C, Bellstedt P, Kirschstein A, Häfner S, Herbst C, Görlach M, Ramachandran R (2014a) Sequential protein NMR assignments in the liquid state via sequential data acquisition. J Magn Reson 239:23–28CrossRefADSGoogle Scholar
  70. Wiedemann C, Bellstedt P, Herbst C, Görlach M, Ramachandran R (2014b) An approach to sequential NMR assignments of proteins: application to chemical shift restraint-based structure prediction. J Biomol NMR 59:211–217CrossRefGoogle Scholar
  71. Wiedemann C, Szambowska A, Häfner S, Ohlenschläger O, Gührs KH, Görlach M (2015) Structure and regulatory role of the C-terminal winged helix domain of the archaeal minichromosome maintenance complex. Nucleic Acids Res 43:2958–2967CrossRefGoogle Scholar
  72. Zawadzka-Kazimierczuk A, Koźmiński W, Šanderová H, Krásný L (2012) High dimensional and high resolution pulse sequences for backbone resonance assignment of intrinsically disordered proteins. J Biomol NMR 52:329–337CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2015

Authors and Affiliations

  • Christoph Wiedemann
    • 1
    • 3
  • Nishit Goradia
    • 1
  • Sabine Häfner
    • 1
  • Christian Herbst
    • 1
    • 2
  • Matthias Görlach
    • 1
  • Oliver Ohlenschläger
    • 1
  • Ramadurai Ramachandran
    • 1
    Email author
  1. 1.Research Group Biomolecular NMR SpectroscopyLeibniz Institute for Age Research, Fritz Lipmann InstituteJenaGermany
  2. 2.Department of Physics, Faculty of ScienceUbon Ratchathani UniversityUbon RatchathaniThailand
  3. 3.Institute of Biochemistry/BiotechnologyMartin Luther University Halle-WittenbergHalle/SalleGermany

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