Journal of Biomolecular NMR

, 51:89 | Cite as

Measurement of 1H–15N and 1H–13C residual dipolar couplings in nucleic acids from TROSY intensities

  • Jinfa Ying
  • Jinbu Wang
  • Alex Grishaev
  • Ping Yu
  • Yun-Xing Wang
  • Ad Bax


Analogous to the recently introduced ARTSY method for measurement of one-bond 1H–15N residual dipolar couplings (RDCs) in large perdeuterated proteins, we introduce methods for measurement of base 13C–1H and 15N–1H RDCs in protonated nucleic acids. Measurements are based on quantitative analysis of intensities in 1H–15N and 13C–1H TROSY-HSQC spectra, and are illustrated for a 71-nucleotide adenine riboswitch. Results compare favorably with those of conventional frequency-based measurements in terms of completeness and convenience of use. The ARTSY method derives the size of the coupling from the ratio of intensities observed in two TROSY-HSQC spectra recorded with different dephasing delays, thereby minimizing potential resonance overlap problems. Precision of the RDC measurements is limited by the signal-to-noise ratio, S/N, achievable in the 2D TROSY-HSQC reference spectrum, and is approximately given by 30/(S/N) Hz for 15N–1H and 65/(S/N) Hz for 13C–1H. The signal-to-noise ratio of both 1H–15N and 1H–13C spectra greatly benefits when water magnetization during the experiments is not perturbed, such that rapid magnetization transfer from bulk water to the nucleic acid, mediated by rapid amino and hydroxyl hydrogen exchange coupled with 1H–1H NOE transfer, allows for fast repetition of the experiment. RDCs in the mutated helix 1 of the riboswitch are compatible with nucleotide-specifically modeled, idealized A-form geometry and a static orientation relative to the helix 2/3 pair, which differs by ca 6° relative to the X-ray structure of the native riboswitch.


ARTSY BEST A-form RNA Quantitative J correlation TROSY RDC RNA DNA 



This work was supported by the Intramural Research Programs of the NIDDK and NCI, NIH, and by the Intramural AIDS-Targeted Antiviral Program of the Office of the Director, NIH.

Supplementary material

10858_2011_9544_MOESM1_ESM.pdf (431 kb)
Supplementary material 1 (PDF 430 kb)


  1. Andersson P, Annila A, Otting G (1998) An alpha/beta-HSQC-alpha/beta experiment for spin-state selective editing of IS cross peaks. J Magn Reson 133:364–367ADSCrossRefGoogle Scholar
  2. Bax A, Grishaev A (2005) Weak alignment NMR: a hawk-eyed view of biomolecular structure. Curr Opin Struct Biol 15:563–570CrossRefGoogle Scholar
  3. Bax A, Griffey RH, Hawkins BL (1983) Correlation of proton and nitrogen-15 chemical shifts by multiple quantum NMR. J Magn Reson 55:301–315CrossRefGoogle Scholar
  4. Bhattacharya A, Revington M, Zuiderweg ERP (2010) Measurement and interpretation of N-15-H-1 residual dipolar couplings in larger proteins. J Magn Reson 203:11–28ADSCrossRefGoogle Scholar
  5. Boisbouvier J, Brutscher B, Simorre JP, Marion D (1999) C-13 spin relaxation measurements in RNA: Sensitivity and resolution improvement using spin-state selective correlation experiments. J Biomol NMR 14:241–252CrossRefGoogle Scholar
  6. Boisbouvier J, Delaglio F, Bax A (2003) Direct observation of dipolar couplings between distant protons in weakly aligned nucleic acids. Proc Natl Acad Sci USA 100:11333–11338ADSCrossRefGoogle Scholar
  7. Bouvignies G, Bernado P, Meier S, Cho K, Grzesiek S, Bruschweiler R, Blackledge M (2005) Identification of slow correlated motions in proteins using residual dipolar and hydrogen-bond scalar couplings. Proc Natl Acad Sci USA 102:13885–13890ADSCrossRefGoogle Scholar
  8. Cho CH, Urquidi J, Singh S, Robinson GW (1999) Thermal offset viscosities of liquid H2O, D2O, and T2O. J Phys Chem B 103:1991–1994CrossRefGoogle Scholar
  9. Clore GM (2000) Accurate and rapid docking of protein–protein complexes on the basis of intermolecular nuclear Overhauser enhancement data and dipolar couplings by rigid body minimization. Proc Natl Acad Sci USA 97:9021–9025ADSCrossRefGoogle Scholar
  10. de Alba E, Tjandra N (2006a) Interference between cross-correlated relaxation and the measurement of scalar and dipolar couplings by quantitative J. J Biomol NMR 35:1–16CrossRefGoogle Scholar
  11. de Alba E, Tjandra N (2006b) On the accurate measurement of amide one-bond N-15-H-1 couplings in proteins: Effects of cross-correlated relaxation, selective pulses and dynamic frequency shifts. J Magn Reson 183:160–165ADSCrossRefGoogle Scholar
  12. 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
  13. Evenas J, Mittermaier A, Yang DW, Kay LE (2001) Measurement of C-13(alpha)-C-13(beta) dipolar couplings in N- 15, C-13, H-2-labeled proteins: Application to domain orientation in maltose binding protein. J Am Chem Soc 123:2858–2864CrossRefGoogle Scholar
  14. Farjon J, Boisbouvier J, Schanda P, Pardi A, Simorre JP, Brutscher B (2009) Longitudinal-Relaxation-Enhanced NMR Experiments for the Study of Nucleic Acids in Solution. J Am Chem Soc 131:8571–8577CrossRefGoogle Scholar
  15. Fitzkee NC, Bax A (2010) Facile measurement of H-1-N-15 residual dipolar couplings in larger perdeuterated proteins. J Biomol NMR 48:65–70CrossRefGoogle Scholar
  16. Freeman R, Kempsell SP, Levitt MH (1980) Radiofrequency pulse sequences which compensate their own imperfections. J Magn Reson 38:453–479CrossRefGoogle Scholar
  17. Geen H, Freeman R (1991) Band-selective radiofrequency pulses. J Magn Reson 93:93–141CrossRefGoogle Scholar
  18. Grishaev A, Ying J, Canny MD, Pardi A, Bax A (2008) Solution structure of tRNA(Val) from refinement of homology model against residual dipolar coupling and SAXS data. J Biomol NMR 42:99–109CrossRefGoogle Scholar
  19. Grishaev A, Yao LS, Ying JF, Pardi A, Bax A (2009) Chemical Shift Anisotropy of Imino N-15 Nuclei in Watson-Crick Base Pairs from Magic Angle Spinning Liquid Crystal NMR and Nuclear Spin Relaxation. J Am Chem Soc 131:9490–9492CrossRefGoogle Scholar
  20. Hansen DF, Vallurupalli P, Kay LE (2008a) Quantifying two-bond (HN)-H-1-(CO)-C-13 and one-bond H-1(alpha)-C-13(alpha) dipolar couplings of invisible protein states by spin-state selective relaxation dispersion NMR spectroscopy. J Am Chem Soc 130:8397–8405CrossRefGoogle Scholar
  21. Hansen DF, Vallurupalli P, Kay LE (2008b) Using relaxation dispersion NMR spectroscopy to determine structures of excited, invisible protein states. J Biomol NMR 41:113–120CrossRefGoogle Scholar
  22. Hwang TL, van Zijl PCM, Garwood M (1997) Broadband adiabatic refocusing without phase distortion. J Magn Reson 124:250–254ADSCrossRefGoogle Scholar
  23. 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
  24. 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 143:184–196ADSCrossRefGoogle Scholar
  25. Kuszewski J, Schwieters C, Clore GM (2001) Improving the accuracy of NMR structures of DNA by means of a database potential of mean force describing base–base positional interactions. J Am Chem Soc 123:3903–3918CrossRefGoogle Scholar
  26. Lescop E, Schanda P, Brutscher B (2007) A set of BEST triple-resonance experiments for time-optimized protein resonance assignment. J Magn Reson 187:163–169ADSCrossRefGoogle Scholar
  27. Levitt MH, Freeman R (1981) Compensation for pulse imperfections in NMR spin-echo experiments. J Magn Reson 43:65–80CrossRefGoogle Scholar
  28. Losonczi JA, Andrec M, Fischer MWF, Prestegard JH (1999) Order matrix analysis of residual dipolar couplings using singular value decomposition. J Magn Reson 138:334–342ADSCrossRefGoogle Scholar
  29. Mantylahti S, Koskela O, Jiang P, Permi P (2010) MQ-HNCO-TROSY for the measurement of scalar and residual dipolar couplings in larger proteins: application to a 557-residue IgFLNa16–21. J Biomol NMR 47:183–194CrossRefGoogle Scholar
  30. Meissner A, Duus JO, Sorensen OW (1997) Spin-state-selective excitation. Application for E.COSY-type measurement of J(HH) coupling constants. J Magn Reson 128:92–97ADSCrossRefGoogle Scholar
  31. Ottiger M, Delaglio F, Bax A (1998) Measurement of J and dipolar couplings from simplified two-dimensional NMR spectra. J Magn Reson 131:373–378ADSCrossRefGoogle Scholar
  32. Palmer AG, Cavanagh J, Wright PE, Rance M (1991) Sensitivity improvement in proton-detected 2-dimensional heteronuclear correlation Nmr-spectroscopy. J Magn Reson 93:151–170CrossRefGoogle Scholar
  33. Permi P, Rosevear PR, Annila A (2000) A set of HNCO-based experiments for measurement of residual dipolar couplings in N-15, C-13, (H-2)-labeled proteins. J Biomol NMR 17:43–54CrossRefGoogle Scholar
  34. Pervushin K, Riek R, Wider G, Wuthrich K (1997) Attenuated T-2 relaxation by mutual cancellation of dipole- dipole coupling and chemical shift anisotropy indicates an avenue to NMR structures of very large biological macromolecules in solution. Proc Natl Acad Sci USA 94:12366–12371ADSCrossRefGoogle Scholar
  35. Pervushin K, Riek R, Wider G, Wuthrich K (1998a) Transverse relaxation-optimized spectroscopy (TROSY) for NMR studies of aromatic spin systems in C-13-labeled proteins. J Am Chem Soc 120:6394–6400CrossRefGoogle Scholar
  36. Pervushin KV, Wider G, Wuthrich K (1998b) Single transition-to-single transition polarization transfer (ST2-PT) in [N15, H1]-TROSY. J Biomol NMR 12:345–348CrossRefGoogle Scholar
  37. Peti W, Meiler J, Bruschweiler R, Griesinger C (2002) Model-free analysis of protein backbone motion from residual dipolar couplings. J Am Chem Soc 124:5822–5833CrossRefGoogle Scholar
  38. Piotto M, Saudek V, Sklenár V (1992) Gradient-tailored excitation for single-quantum NMR spectroscopy of aqueous sloutions. J Biomol NMR 2:661–665CrossRefGoogle Scholar
  39. Prestegard JH, Al-Hashimi HM, Tolman JR (2000) NMR structures of biomolecules using field oriented media and residual dipolar couplings. Q Rev Biophys 33:371–424CrossRefGoogle Scholar
  40. Schanda P, Van Melckebeke H, Brutscher B (2006) Speeding up three-dimensional protein NMR experiments to a few minutes. J Am Chem Soc 128:9042–9043CrossRefGoogle Scholar
  41. Serganov A, Patel DJ (2007) Ribozymes, riboswitches and beyond: regulation of gene expression without proteins. Nat Rev Genet 8:776–790CrossRefGoogle Scholar
  42. Skrynnikov NR, Kay LE (2000) Assessment of molecular structure using frame-independent orientational restraints derived from residual dipolar couplings. J Biomol NMR 18:239–252CrossRefGoogle Scholar
  43. Skrynnikov NR, Goto NK, Yang DW, Choy WY, Tolman JR, Mueller GA, Kay LE (2000) Orienting domains in proteins using dipolar couplings measured by liquid-state NMR: Differences in solution and crystal forms of maltodextrin binding protein loaded with beta-cyclodextrin. J Mol Biol 295:1265–1273CrossRefGoogle Scholar
  44. Tjandra N, Bax A (1997a) Direct measurement of distances and angles in biomolecules by NMR in a dilute liquid crystalline medium. Science 278:1111–1114ADSCrossRefGoogle Scholar
  45. Tjandra N, Bax A (1997b) Measurement of dipolar contributions to (1)J(CH) splittings from magnetic-field dependence of J modulation in two-dimensional NMR spectra. J Magn Reson 124:512–515ADSCrossRefGoogle Scholar
  46. Tolbert BS, Miyazaki Y, Barton S, Kinde B, Starck P, Singh R, Bax A, Case DA, Summers MF (2010) Major groove width variations in RNA structures determined by NMR and impact of C-13 residual chemical shift anisotropy and H-1-C-13 residual dipolar coupling on refinement. J Biomol NMR 47:205–219CrossRefGoogle Scholar
  47. Tolman JR (2002) A novel approach to the retrieval of structural and dynamic information from residual dipolar couplings using several oriented media in biomolecular NMR spectroscopy. J Am Chem Soc 124:12020–12030CrossRefGoogle Scholar
  48. Tolman JR, Ruan K (2006) NMR residual dipolar couplings as probes of biomolecular dynamics. Chem Rev 106:1720–1736CrossRefGoogle Scholar
  49. Vallurupalli P, Hansen DF, Kay LE (2008) Structures of invisible, excited protein states by relaxation dispersion NMR spectroscopy. Proc Natl Acad Sci USA 105:11766–11771ADSCrossRefGoogle Scholar
  50. van Ingen H, Korzhnev DM, Kay LE (2009) An Analysis of the Effects of H-1(N)-H-1(N) Dipolar Couplings on the Measurement of Amide Bond Vector Orientations in Invisible Protein States by Relaxation Dispersion NMR. J Phys Chem B 113:9968–9977CrossRefGoogle Scholar
  51. Vijayan V, Zweckstetter M (2005) Simultaneous measurement of protein one-bond residual dipolar couplings without increased resonance overlap. J Magn Reson 174:245–253ADSCrossRefGoogle Scholar
  52. Wang JB, Zuo XB, Yu P, Xu H, Starich MR, Tiede DM, Shapiro BA, Schwieters CD, Wang YX (2009) A Method for Helical RNA Global Structure Determination in Solution Using Small-Angle X-Ray Scattering and NMR Measurements. J Mol Biol 393:717–734CrossRefGoogle Scholar
  53. Yang DW, Venters RA, Mueller GA, Choy WY, Kay LE (1999) TROSY-based HNCO pulse sequences for the measurement of (HN)-H- 1-N-15, N-15-(CO)-C-13, (HN)-H-1-(CO)-C-13, (CO)-C-13-C- 13(alpha) and (HN)-H-1-C-13(alpha) dipolar couplings in N-15, C-13, H-2-labeled proteins. J Biomol NMR 14:333–343CrossRefGoogle Scholar
  54. Yao L, Vogeli B, Torchia DA, Bax A (2008) Simultaneous NMR study of protein structure and dynamics using conservative mutagenesis. J Phys Chem B 112:6045–6056CrossRefGoogle Scholar
  55. Yao LS, Ying JF, Bax A (2009) Improved accuracy of N-15-H-1 scalar and residual dipolar couplings from gradient-enhanced IPAP-HSQC experiments on protonated proteins. J Biomol NMR 43:161–170CrossRefGoogle Scholar
  56. Ying JF, Grishaev AE, Bax A (2006) Carbon-13 chemical shift anisotropy in DNA bases from field dependence of solution NMR relaxation rates. Magn Reson Chem 44:302–310CrossRefGoogle Scholar
  57. Ying JF, Chill JH, Louis JM, Bax A (2007a) Mixed-time parallel evolution in multiple quantum NMR experiments: sensitivity and resolution enhancement in heteronuclear NMR. J Biomol NMR 37:195–204CrossRefGoogle Scholar
  58. Ying JF, Grishaev A, Latham MP, Pardi A, Bax A (2007b) Magnetic field induced residual dipolar couplings of imino groups in nucleic acids from measurements at a single magnetic field. J Biomol NMR 39:91–96CrossRefGoogle Scholar
  59. Zhang Q, Sun XY, Watt ED, Al-Hashimi HM (2006) Resolving the motional modes that code for RNA adaptation. Science 311:653–656ADSCrossRefGoogle Scholar
  60. Zhang Q, Stelzer AC, Fisher CK, Al-Hashimi HM (2007) Visualizing spatially correlated dynamics that directs RNA conformational transitions. Nature 450:1263–1267ADSCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. (outside the USA) 2011

Authors and Affiliations

  • Jinfa Ying
    • 1
  • Jinbu Wang
    • 2
  • Alex Grishaev
    • 1
  • Ping Yu
    • 2
  • Yun-Xing Wang
    • 2
  • Ad Bax
    • 1
  1. 1.Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney DiseasesNational Institutes of HealthBethesdaUSA
  2. 2.Structural Biophysics LaboratoryNational Cancer Institute, National Institutes of HealthFrederickUSA

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