Journal of Biomolecular NMR

, Volume 52, Issue 2, pp 115–126 | Cite as

Efficient sequential assignments in proteins with reduced dimensionality 3D HN(CA)NH

  • Kousik Chandra
  • Garima Jaipuria
  • Divya Shet
  • Hanudatta S. AtreyaEmail author


We present reduced dimensionality (RD) 3D HN(CA)NH for efficient sequential assignment in proteins. The experiment correlates the 15N and 1H chemical shift of a residue (‘i’) with those of its immediate N-terminal (i − 1) and C-terminal (i + 1) neighbors and provides four-dimensional chemical shift correlations rapidly with high resolution. An assignment strategy is presented which combines the correlations observed in this experiment with amino acid type information obtained from 3D CBCA(CO)NH. By classifying the 20 amino acid types into seven distinct categories based on 13Cβ chemical shifts, it is observed that a stretch of five sequentially connected residues is sufficient to map uniquely on to the polypeptide for sequence specific resonance assignments. This method is exemplified by application to three different systems: maltose binding protein (42 kDa), intrinsically disordered domain of insulin-like growth factor binding protein-2 and Ubiquitin. Fast data acquisition is demonstrated using longitudinal 1H relaxation optimization. Overall, 3D HN(CA)NH is a powerful tool for high throughput resonance assignment, in particular for unfolded or intrinsically disordered polypeptides.


Sequence specific resonance assignment Reduced dimensionality NMR Protein structure GFT NMR 



The facilities provided by NMR Research Centre at IISc and National facility for high-field NMR at Tata Institute of Fundamental Research (TIFR; Mumbai) supported by Department of Science and Technology (DST), India is gratefully acknowledged. HSA acknowledges support from DST-SERC and Department of Biotechnology (DBT) research awards. GJ acknowledges fellowship from Council of Scientific and Industrial Research (CSIR), India. We thank Dr. John Cort, Pacific Northwest National Laboratory, for providing the Ubiquitin plasmid and Prof. R. V. Hosur, TIFR for providing the MBP sample.

Supplementary material

10858_2011_9598_MOESM1_ESM.pdf (332 kb)
Supplementary material 1 (PDF 331 kb)


  1. Atreya HS, Chary KVR (2002) Automated NMR assignments of proteins for high throughput structure determination: TATAPRO II. Curr Sci 83:1372–1376Google Scholar
  2. Atreya HS, Szyperski T (2004) G-matrix Fourier transform NMR spectroscopy for complete protein resonance assignment. Proc Natl Acad Sci 101:9642–9647ADSCrossRefGoogle Scholar
  3. Atreya HS, Szyperski T (2005) Rapid NMR data collection. Methods Enzymol 394:78–108CrossRefGoogle Scholar
  4. Atreya HS, Sahu SC, Chary KVR, Govil G (2000) A tracked approach for automated NMR assignments in proteins (TATAPRO). J Biomol NMR 17:125–136CrossRefGoogle Scholar
  5. Baran MC, Huang YJ, Moseley HNB, Montelione GT (2004) Automated analysis of protein NMR assignments and structures. Chem Rev 104:3541–3555CrossRefGoogle Scholar
  6. Bartels C, Xia TH, Billeter M, Güntert P, Wüthrich K (1995) The program XEASY for computer-supported Nmr spectral-analysis of biological macromolecules. J Biomol NMR 6:1–10CrossRefGoogle Scholar
  7. Bracken C, Palmer AG, Cavanagh J (1997) (H)N(COCA)NH and HN(COCA)NH experiments for 1H–15N backbone assignments in C-13/N-15-labeled proteins. J Biomol NMR 9:94–100CrossRefGoogle Scholar
  8. Cavanagh J, Fairbrother WJ, Palmer AG, Rance M, Skelton NJ (2007) Protein NMR spectroscopy. Academic Press, San DiegoGoogle Scholar
  9. Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, Bax A (1995) NMRPIPE—a multidimensional spectral processing system based on Unix pipes. J Biomol NMR 6:277–293CrossRefGoogle Scholar
  10. Diercks T, Daniels M, Kaptein R (2005) Extended flip-back schemes for sensitivity enhancement in multidimensional HSQC-type out-and-back experiments. J Biomol NMR 33:243–259CrossRefGoogle Scholar
  11. Dyson HJ, Wright PE (2005) Intrinsically unstructured proteins and their functions. Nat Rev Mol Cell Biol 6:197–208CrossRefGoogle Scholar
  12. Fiorito F, Hiller S, Wider G, Wüthrich K (2006) Automated resonance assignment of proteins: 6D APSY-NMR. J Biomol NMR 35:27–37CrossRefGoogle Scholar
  13. Frueh DP, Sun ZY, 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 128:5757–5763CrossRefGoogle Scholar
  14. Frueh DP, Arthanari H, Koglin A, Walsh CT, Wagner G (2009) A double TROSY hNCAnH experiment for efficient assignment of large and challenging proteins. J Am Chem Soc 131:12880–12881CrossRefGoogle Scholar
  15. 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 129:10823–10828CrossRefGoogle Scholar
  16. Ikegami T, Sato S, Walchli M, Kyogoku Y, Shirakawa M (1997) An efficient HN(CA)NH pulse scheme for triple-resonance 4D correlation of sequential amide protons and nitrogens-15 in deuterated proteins. J Magn Reson 124:214–217ADSCrossRefGoogle Scholar
  17. Kim S, Szyperski T (2003) GFT NMR, a new approach to rapidly obtain precise high-dimensional NMR spectral information. J Am Chem Soc 125:1385–1393CrossRefGoogle Scholar
  18. Krishnarjuna B, Jaipuria G, Thakur A, D’Silva P, Atreya HS (2011) Amino acid selective unlabeling for sequence specific resonance assignments in proteins. J Biomol NMR 49:39–51CrossRefGoogle Scholar
  19. Liu GH, Shen Y, Atreya HS, Parish D, Shao Y, Sukumaran DK, Xiao R, Yee A, Lemak A, Bhattacharya A, Acton TA, Arrowsmith CH, Montelione GT, Szyperski T (2005) NMR data collection and analysis protocol for high-throughput protein structure determination. Proc Natl Acad Sci 102:10487–10492ADSCrossRefGoogle Scholar
  20. Ohki SY, Kainosho M (2008) Stable isotope labeling methods for protein NMR spectroscopy. Prog Nucl Magn Reson Spectrosc 53:208–226CrossRefGoogle Scholar
  21. 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 20:135–147CrossRefGoogle Scholar
  22. Pervushin K, Vogeli B, Eletsky A (2002) Longitudinal 1H relaxation optimization in TROSY NMR spectroscopy. J Am Chem Soc 124:12898–12902CrossRefGoogle Scholar
  23. Shirakawa M, Walchli M, Shimizu M, Kyogoku Y (1995) The use of heteronuclear cross-polarisation for backbone assignment of H-2-labeled, N-15-labeled and C-13-labeled proteins—a pulse scheme for triple-resonance 4D correlation of sequential amide protons and N-15. J Biomol NMR 5:323–326CrossRefGoogle Scholar
  24. Swain M, Slomiany MG, Rosenzweig SA, Atreya HS (2010) High-yield bacterial expression and structural characterization of recombinant human insulin-like growth factor binding protein-2. Arch Biochem Biophys 501:195–200CrossRefGoogle Scholar
  25. Szyperski T, Atreya HS (2006) Principles and applications of GFT projection NMR spectroscopy. Magn Reson Chem 44:S51–S60CrossRefGoogle Scholar
  26. Szyperski T, Braun D, Fernandez C, Bartels C, Wüthrich K (1995) A novel reduced-dimensionality triple resonance experiment for efficient polypeptide backbone assignment, 3D COHNNCA. J Magn Reson B108:197–203Google Scholar
  27. Szyperski T, Braun D, Banecki B, Wüthrich K (1996) Useful information from axial peak magnetization in projected NMR experiments. J Am Chem Soc 118:8146–8147CrossRefGoogle Scholar
  28. Szyperski T, Yeh DC, Sukumaran DK, Moseley HNB, Montelione GT (2002) Reduced-dimensionality NMR spectroscopy for high-throughput protein resonance assignment. Proc Natl Acad Sci 99:8009–8014ADSCrossRefGoogle Scholar
  29. Takeuchi K, Gal M, Takahashi H, Shimada I, Wagner G (2011) HNCA-TOCSY-CANH experiments with alternate (13)C-(12)C labeling: a set of 3D experiment with unique supra-sequential information for mainchain resonance assignment. J Biomol NMR 49:17–26CrossRefGoogle Scholar
  30. Tugarinov V, Hwang PM, Kay LE (2004) Nuclear magnetic resonance spectroscopy of high-molecular-weight proteins. Ann Rev Biochem 73:107–146CrossRefGoogle Scholar
  31. Weisemann R, Rüterjans H, Bermel W (1993) 3D triple-resonance NMR techniques for the sequential assignment of NH and N-15 resonances in N-15-labeled and C-13-labeled proteins. J Biomol NMR 3:113–120CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Kousik Chandra
    • 1
  • Garima Jaipuria
    • 1
    • 2
  • Divya Shet
    • 1
  • Hanudatta S. Atreya
    • 1
    Email author
  1. 1.NMR Research CentreIndian Institute of ScienceBangaloreIndia
  2. 2.Solid State and Structural Chemistry UnitIndian Institute of ScienceBangaloreIndia

Personalised recommendations