Journal of Biomolecular NMR

, Volume 47, Issue 3, pp 183–194

MQ-HNCO-TROSY for the measurement of scalar and residual dipolar couplings in larger proteins: application to a 557-residue IgFLNa16-21

  • Sampo Mäntylahti
  • Outi Koskela
  • Pengju Jiang
  • Perttu Permi


We describe a novel pulse sequence, MQ-HNCO-TROSY, for the measurement of scalar and residual dipolar couplings between amide proton and nitrogen in larger proteins. The experiment utilizes the whole 2TN polarization transfer delay for labeling of 15N chemical shift in a constant time manner, which efficiently doubles the attainable resolution in 15N dimension with respect to the conventional HNCO-TROSY experiment. In addition, the accordion principle is employed for measuring (J + D)NHs, and the multiplet components are selected with the generalized version of the TROSY scheme introduced by Nietlispach (J Biomol NMR 31:161–166, 2005). Therefore, cross peak overlap is diminished while the time period during which the 15N spin is susceptible to fast transverse relaxation associated with the anti-TROSY transition is minimized per attainable resolution unit. The proposed MQ-HNCO-TROSY scheme was employed for measuring RDCs in high molecular weight protein IgFLNa16-21 of 557 residues, resulting in 431 experimental RDCs. Correlations between experimental and back-calculated RDCs in individual domains gave relatively low Q-factors (0.19–0.39), indicative of sufficient accuracy that can be obtained with the proposed MQ-HNCO-TROSY experiment in high molecular weight proteins.


Dipolar couplings HNCO NMR Proteins Scalar couplings TROSY 


  1. Abrogast L, Majumdar A, Tolman JR (2010) HNCO-based measurement of one-bond amide 15N–1H couplings with optimized precision. J Biomol NMR 46:175–189CrossRefGoogle Scholar
  2. Andersson P, Annila A, Otting G (1998) An α/β-HSQC-α/β experiment for spin-state selective editing of IS cross peaks. J Magn Reson 133:364–367CrossRefADSGoogle Scholar
  3. Annila A, Permi P (2004) Weakly aligned biological macromolecules in dilute aqueous liquid crystals. Concepts Magn Reson 23A:22–37CrossRefGoogle Scholar
  4. Bax A, Kontaxis G, Tjandra N (2001) Dipolar couplings in macromolecular structure determination. Methods Enzymol 339:127–174CrossRefGoogle Scholar
  5. Blackledge M (2005) Recent progress in the study of biomolecular structure and dynamics in solution from residual dipolar couplings. Prog Nucl Magn Reson Spectr 46:23–61CrossRefGoogle Scholar
  6. Bodenhausen G, Ernst RR (1981) The accordion experiment, a simple approach to 3-dimensional NMR spectroscopy. J Magn Reson 45:367–373Google Scholar
  7. Bouvignies G, Markwick PRL, Blackledge M (2007) Simultaneous definition of high resolution protein structure and backbone conformational dynamics using NMR residual dipolar couplings. ChemPhysChem 8:1901–1909CrossRefGoogle Scholar
  8. Düx P, Whitehead B, Boelens R, Kaptein R, Vuister GW (1999) Measurement of 15N–1H coupling constants in uniformly 15N-labeled proteins: application to the photoactive yellow protein. J Biomol NMR 10:301–306CrossRefGoogle Scholar
  9. Fischer MW, Losonczi JA, Weaver JL, Prestegard JH (1999) Domain orientation and dynamics in multidomain protein from residual dipolar couplings. Biochemistry 38:9013–9022CrossRefGoogle Scholar
  10. Fredriksson K, Louhivuori M, Permi P, Annila A (2004) On the interpretation of residual dipolar couplings as reporters of molecular dynamics. J Am Chem Soc 126:12646–12650CrossRefGoogle Scholar
  11. Hansen MR, Mueller L, Pardi A (1998) Tunable alignment of macromolecules by filamentous phage yields dipolar coupling interactions. Nat Struct Biol 5:1065–1074CrossRefGoogle Scholar
  12. Heikkinen S, Aitio H, Permi P, Folmer R, Lappalainen K, Kilpeläinen I (1999) J-multiplied HSQC (MJ-HSQC): a new method for measuring 3J(HNHα) couplings in 15N-labeled proteins. J Magn Reson 137:243–246CrossRefADSGoogle Scholar
  13. Heikkinen OK, Ruskamo S, Konarev PV, Svergun DI, Iivanainen T, Heikkinen SM, Permi P, Koskela H, Kilpeläinen I, Ylänne J (2009) Atomic structures of two novel immunoglobulin-like domain pairs in the actin cross-linking protein filamin. J Biol Chem 284:25450–25458CrossRefGoogle Scholar
  14. Hu K, Doucleff M, Clore GM (2009) Using multiple quantum-coherence to increase the 15N resolution in a three-dimensional TROSY-HNCO experiment for accurate PRE and RDC measurements. J Magn Reson 200:173–177CrossRefADSGoogle Scholar
  15. Kay LE, Keifer P, Saarinen T (1992) Pure absorption gradient-enhanced heteronuclear single-quantum correlation spectroscopy with improved sensitivity. J Am Chem Soc 114:10663–10665CrossRefGoogle Scholar
  16. Kontaxis G, Clore GM, Bax A (2000) Evaluation of cross-correlation effects and measurement of one-bond couplings in proteins with short transverse relaxation times. J Magn Reson 143:184–196CrossRefADSGoogle Scholar
  17. Lad Y, Kiema T, Jiang P, Pentikäinen O, Coles CH, Campbell ID, Calderwood DA, Ylänne J (2007) Structure of three tandem filamin domains reveals auto-inhibition of ligand binding. EMBO J 26:3993–4004CrossRefGoogle Scholar
  18. Lakomek NA, Carlomagno T, Becker S, Griesinger C, Meiler J (2006) A thorough dynamic interpretation of residual dipolar couplings in ubiquitin. J Biomol NMR 34:101–115CrossRefGoogle Scholar
  19. Lerche MH, Meissner A, Poulsen FM, Sørensen OW (1999) Pulse sequences for measurement of one-bond (15)N-(1)H coupling constants in the protein backbone. J Magn Reson 140:259–263CrossRefADSGoogle Scholar
  20. Losonczi JA, Andrec M, Fischer MWF, Prestegard JH (1999) Order matrix analysis of residual dipolar couplings using singular-value decomposition. J Magn Reson 138:334–342CrossRefADSGoogle Scholar
  21. Madsen JC, Sørensen OW, Sørensen P, Poulsen FM (1993) Improved pulse sequences for measuring coupling constants in 13C, 15N-labeled proteins. J Biomol NMR 3:239–244CrossRefGoogle Scholar
  22. Marion D, Ikura M, Tschudin R, Bax A (1989) Rapid recording of 2D NMR-spectra without phase cycling—application to the study of hydrogen-exchange in proteins. J Magn Reson 85:393–399Google Scholar
  23. McCoy MA, Mueller L (1992) Selective shaped pulse decoupling in NMR: homonuclear [13C]carbonyl decoupling. J Am Chem Soc 114:2108–2112CrossRefGoogle Scholar
  24. Meissner A, Duus JO, Sørensen OW (1997) Integration of spin-state-selective excitation into 2D NMR correlation experiments with the heteronuclear ZQ/DQ π rotations for 1JXH-resolved E.COSY-type measurement of heteronuclear coupling constants in proteins. J Biomol NMR 10:89–94CrossRefGoogle Scholar
  25. Nietlispach D (2005) Suppression of anti-TROSY lines in a sensitivity-enhanced gradient selection TROSY scheme. J Biomol NMR 31:161–166CrossRefGoogle Scholar
  26. Ottiger M, Delaglio F, Bax A (1998) Measurement of J and dipolar couplings from simplified two-dimensional NMR spectra. J Magn Reson 131:373–378CrossRefADSGoogle Scholar
  27. Pääkkönen K, Sorsa T, Drakenberg T, Pollesello P, Tilgmann C, Permi P, Heikkinen S, Kilpeläinen I, Annila A (2000) Conformations of the regulatory domain of cardiac troponin C examined by residual dipolar couplings. Eur J Biochem 267:6665–6672CrossRefGoogle Scholar
  28. Permi P (2002) A spin-state-selective experiment for measuring heteronuclear one-bond and homonuclear two-bond couplings from an HSQC-type spectrum. J Biomol NMR 22:27–35CrossRefGoogle Scholar
  29. Permi P (2003) Measurement of residual dipolar couplings from 1Hα to 13Cα and 15N using a simple HNCA-based experiment. J Biomol NMR 27:341–349CrossRefGoogle Scholar
  30. Permi P, Rosevear PR, Annila A (2000a) A set of HNCO-based experiments for measurement of residual dipolar couplings in 15N, 13C, (2H) labeled proteins. J Biomol NMR 17:43–54CrossRefGoogle Scholar
  31. Permi P, Kilpeläinen I, Annila A (2000b) Determination of backbone angle ψ in proteins using a TROSY-based α/β-HN(CO)CA-J experiment. J Magn Reson 146:255–259CrossRefADSGoogle Scholar
  32. Pervushin K, Billeter M, Siegal G, Wüthrich K (1997) Attenuated T2 relaxation by mutual cancellation of dipole–dipole coupling and chemical shift anisotropy indicates an avenue to NMR structures very large biological macromolecules in solution. Proc Natl Acad Sci U S A 94:12366–12371CrossRefADSGoogle Scholar
  33. Prestegard JH, Al-Hashimi HM, Tolman JR (2000) NMR structures of biomolecules using field oriented media and residual dipolar couplings. Quart Rev Biophys 33:371–424CrossRefGoogle Scholar
  34. Puttonen E, Tossavainen H, Permi P (2006) Simultaneous determination of one- and two-bond scalar and residual dipolar couplings between 13C′, 13Cα, and 15N spins in proteins. Magn Reson Chem 44:168–176CrossRefGoogle Scholar
  35. Salzmann M, Pervushin K, Wider G, Senn H, Wüthrich K (1998) TROSY in triple-resonance experiments: new perspectives for sequential NMR assignment of large proteins. Proc Natl Acad Sci U S A 95:13585–13590CrossRefADSGoogle Scholar
  36. Tjandra N, Bax A (1997) Direct measurement of distances and angles in biomolecules by NMR in a dilute liquid crystalline medium. Science 278:1111–1114CrossRefADSGoogle Scholar
  37. Tolman JR, Ruan K (2006) NMR residual dipolar couplings as probes of biomolecular dynamics. Chem Rev 106:1720–1736CrossRefGoogle Scholar
  38. Tugarinov V, Kay LE (2003) Quantitative NMR studies of high molecular weight proteins: application to domain orientation and ligand binding in the 723 residue enzyme malate synthase G. J Mol Biol 327:1121–1133CrossRefGoogle Scholar
  39. Wang AC, Bax A (1995) Reparametrization of the Karplus relation for 3J(Hα-N) and 3J(HN-C′) in peptides from uniformly 13C/15N-enriched human ubiquitin. J Am Chem Soc 117:1810–1813CrossRefGoogle Scholar
  40. Weigelt J (1998) Single scan, sensitivity- and gradient-enhanced TROSY for multidimensional NMR experiments. J Am Chem Soc 120:10778–10779CrossRefGoogle Scholar
  41. Yang DW, Kay LE (1999) Improved 1HN-detected triple resonance TROSY-based experiments. J Biomol NMR 13:3–10CrossRefGoogle Scholar
  42. Yang DW, Venters RA, Mueller GA, Choy WY, Kay LE (1999) TROSY-based HNCO pulse sequences for the measurement of 1HN-15N, 15N–13CO, 1HN-13CO, 13CO-13Ca and 1HN-13Ca dipolar couplings in 15N, 13C, 2H-labeled proteins. J Biomol NMR 14:333–343CrossRefGoogle Scholar
  43. Zweckstetter M, Bax A (2000) Prediction of sterically induced alignment in a dilute liquid crystalline phase: aid to protein structure determination by NMR. J Am Chem Soc 122:3791–3792CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  • Sampo Mäntylahti
    • 1
  • Outi Koskela
    • 2
  • Pengju Jiang
    • 3
  • Perttu Permi
    • 1
  1. 1.NMR Laboratory, Program in Structural Biology and Biophysics, Institute of Biotechnology/NMR LaboratoryUniversity of HelsinkiHelsinkiFinland
  2. 2.Laboratory of Organic Chemistry, Department of ChemistryUniversity of HelsinkiHelsinkiFinland
  3. 3.Biochemistry DepartmentUniversity of OxfordOxfordUK

Personalised recommendations