Double and Triple Resonance NMR Methods for Protein Assignment

  • Brian Whitehead
  • C. Jeremy Craven
  • Jonathan P. Waltho
Part of the Methods in Molecular Biology™ book series (MIMB, volume 60)


Assignment of the spin systems in protein NMR spectra is an essential step in solution structure determination. The development of two-dimensional (2D) NMR experiments during the 1970s and 1980s allowed complex overlapped spectra to be unambiguously assigned for the first time, and since the mid-1980s over 100 protein solution structures have been determined by NMR. The techniques used for 1H assignment in unlabeled proteins are covered in several texts, including a previous volume in this series (1, 2, 3, 4), and will not be discussed further here. 1H homonuclear assignment strategies are usually insufficient for proteins with mol wt >10 kDa, however, and the limitations of the experiments will be outlined in the following discussion.


Magnetization Transfer Assignment Strategy Amide Proton Sequential Assignment Heteronuclear Single Quantum Coherence 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


  1. 1.
    Wuthrich, K. (1986) NMR of Proteins and Nucleic Acids, Wiley, New York.Google Scholar
  2. 2.
    Redfield, C. (1993) Resonance assignment strategies for small proteins, in NMR of Macromolecules. (Roberts, G.C.K., ed.), Oxford University Press, Oxford, pp. 71–99.Google Scholar
  3. 3.
    Neuhaus, D. and Evans, P. A. (1993) Structural studies of proteins in solution using protein NMR, in Methods in Molecular Biology vol. 17, Spectroscopic Methods and Analyses NMR, Mass Spectrometry and Metalloprotein Techniques. (Jones, C., Mulloy, B., and Thomas, A. H., eds.), Humana, Totowa, NJ, pp. 15–67.Google Scholar
  4. 4.
    Basus, V. L. (1989) Proton nuclear magnetic resonance assignment, in Methods in Enzymology, vol. 177, Nuclear Magnetic Resonance part B. (James, T. L. and Oppenheimer, N. J., eds.), Academic, New York, pp. 132–149.CrossRefGoogle Scholar
  5. 5.
    Lipari, G. and Szabo, A. (1982) Model-free approach to the interpretation of nuclear magnetic resonance relaxation in macromolecules 1 Theory and range of validity. J. Am. Chem. Soc. 104, 4546–4559.CrossRefGoogle Scholar
  6. 6.
    Edison, A. S., Markley, J. L., and Weinhold, F. (1994) Calculations of one-, two-, and three-bond nuclear spin-spin couplings in a model peptide and correlations with experimental data. J. Biomol. NMR 4, 519–542.PubMedCrossRefGoogle Scholar
  7. 7.
    Edison, A. S., Abildgaard, F., Westler, W. M., Mooberry, E. S., and Markley, J. L. (1994) Practical introduction to theory and implementation of multinuclear, multi-dimensional nuclear magnetic resonance experiments, in Methods in Enzymology, vol. 239, Nuclear Magnetic Resonance Part C (James, T. L. and Oppenheimer, N. J., eds.), Academic, New York, pp. 3–79.CrossRefGoogle Scholar
  8. 8.
    Bax, A., Ikura, M., Kay, L., Torchia, D. A., and Tschudin, R. (1990) Comparison of different modes of two-dimensional reverse-correlation NMR for the study of proteins. J. Magn. Reson. 86, 304–318.Google Scholar
  9. 9.
    Norwood, T. J., Boyd, J., Heritage, J. E., Soffe, N., and Campbell, I. D. (1990) Comparison of techniques for 1H-detected 1H-15N spectroscopy. J. Magn. Reson. 87, 488–501.Google Scholar
  10. 10.
    Bodenhausen, G. and Ruben, D. J. (1980) Natural abundance nitrogen-15 NMR by enhanced heteronuclear spectroscopy. Chem. Phys. Lett. 69, 185–189.CrossRefGoogle Scholar
  11. 11.
    Martin, J. R., Jerala, R., Kroon-Zitko, L., Zerovnik, E., Turk, V., and Waltho, J. (1994) Structural characterisation of human stefin A in solution and implications for binding to cysteine proteases. Eur. J. Biochem. 225, 1181–1194.PubMedCrossRefGoogle Scholar
  12. 12.
    Marion, D., Driscoll, P. C., Kay, L. E., Wingfield, P. T., Bax, A., Gronenborn, A. M., and Clore, G. M. (1989) Overcoming the overlap problem in the assignment of 1H NMR spectra of larger proteins by use of three-dimensional heteronuclear 1H-15N Hartmann-Hahn-multiple quantum coherence and Nuclear Overhauser-multiple quantum coherence spectroscopy. Application to Interleukin 1 β. Biochemistry 28, 6150–6156.PubMedCrossRefGoogle Scholar
  13. 13.
    Carr, M. D., Birdsall, B., Frenkiel, T. A., Bauer, C. J., Jimenez-Barbero, J., Polshakov, V. I., McCormick, J. E., Roberts, G. C. K., and Feeney, J. (1991) Dihydrofolate reductase: Sequential resonance assignments using 2D and 3D NMR and secondary structure determination in solution. Biochemistry 30, 6330–6341.PubMedCrossRefGoogle Scholar
  14. 14.
    Ikura, M., Bax, A., Clore, G. M., and Gronenborn, A. M. (1990) Detection of Nuclear Overhauser effects between degenerate amide proton resonances by heteronuclear three-dimensional NMR. J. Am. Chem. Soc. 112, 9020–9022.CrossRefGoogle Scholar
  15. 15.
    Stockman, B. J., Euvrard, A., Kloosterman, D. A., Scahill, T. A., and Swensom, R. P. (1993) 1H and 15N resonance assignments and solution secondary structure of oxidised Desulfovibrio vulgaris flavodoxin determined by heteronuclear three-dimensional NMR spectroscopy. J. Biomol. NMR 3, 133–149.PubMedGoogle Scholar
  16. 16.
    Wijmenga, S. S., Hallenga, K., and Hilbers, C. W. (1989) A three-dimensional heteronuclear multiple-quantum coherence homonuclear Hartman-Hahn experiment. J. Magn. Reson. 84, 634–642.Google Scholar
  17. 17.
    Fesik, S. W. and Zuiderweg, E. R. P. (1988) Heteronuclear three-dimensional NMR spectroscopy. A strategy for the simplification of homonuclear two-dimensional NMR spectra. J. Magn. Reson. 78, 588–593.Google Scholar
  18. 18.
    Gronenborn, A. M., Bax, A., Wingfield, P. T., and Clore, G. M. (1989) A powerful method of sequential proton assignment in proteins using relayed 15N–1H multiple quantum coherence spectroscopy. FEBS Lett. 243, 93–98.PubMedCrossRefGoogle Scholar
  19. 19.
    Gronenborn, A. M., Wingfield, P. T., and Clore, G. in (1989) Determination of the secondary structure of the DNA binding protein Ner from Phage Mu using 1H homonuclear and 15N–1H heteronuclear NMR spectroscopy. Biochemistry 28, 5081–5089.PubMedCrossRefGoogle Scholar
  20. 20.
    Keeler, J., Clowes, R. T., Davis, A. L., and Laue, E. D. (1994) Pulsed-field gradients: theory and practice, in Methods in Enzymology, vol. 239, Nuclear Magnetic Resonance part C. (James, T. L. and Oppenheimer, N. J., eds.), Academic, New York, pp. 145–207.CrossRefGoogle Scholar
  21. 21.
    Kay, L., Ikura, M., Tschudin, R., and Bax, A. (1990) Three-dimensional triple-resonance NMR spectroscopy of isotopically enriched proteins. J. Magn. Reson. 89, 496–514.Google Scholar
  22. 22.
    Ikura, M., Kay, L. E., and Bax, A. (1990) A novel approach for sequential assignment of larger proteins: Heteronuclear triple-resonance three-dimensional NMR spectroscopy. Application to calmodulin. Biochemistry 29, 4659–4667.PubMedCrossRefGoogle Scholar
  23. 23.
    Farmer, B. T., Venters, R. A., Spacer, L. D., Wittekind, M. G., and Muller, L. (1992) A refocused and optimized HNCA: Increased sensitivity and resolution in large macromolecules. J. Biomol. NMR 2, 195–202.PubMedCrossRefGoogle Scholar
  24. 24.
    Grzesiek, S. and Bax, A. (1992) Improved 3D triple-resonance NMR techniques. J. Magn. Reson. 96, 432–440.Google Scholar
  25. 25.
    Bax, A. and Ikura, M. (1991) An efficient 3D NMR technique for correlating the proton and 15N backbone amide resonances with the α-carbon of the preceding residue in uniformly 15N/13C enriched proteins. J. Biomol. NMR 1, 99–104.PubMedCrossRefGoogle Scholar
  26. 26.
    Kay, L. E., Ikura, M., and Bax, A. (1991) The design and optimization of complex NMR experiments. Application to a triple-resonance pulse scheme correlating Hα, NH and 15N chemical shifts in 15N-13C-labeled proteins. J. Magn. Reson. 91, 84–92.Google Scholar
  27. 27.
    Seip, S., Balbach, J., and Kessler, H. (1992) An improved technique for correlating backbone amide protons with 15N and Hα protons (HN(CA)H) in isotopically enriched proteins. J. Magn. Reson. 100, 406–410.Google Scholar
  28. 28.
    Clubb, R. T., Thanabal, V., and Wagner, G. (1992) A new 3D HN(CA)HA experiment for obtaining fingerprint HN-Hα cross peaks in 15N-and 13C-labelled proteins. J. Biomol. NMR 2, 203–210.PubMedCrossRefGoogle Scholar
  29. 29.
    Fogh, R. H., Schipper, D., Boelens, R., and Kaptein, R. (1994) 1H, 13C and 151N NMR backbone assignments of the 269-residue serine protease PB92 from Bacillus alcalophil J. Biomol. NMR 4, 123–128.CrossRefGoogle Scholar
  30. 30.
    Clubb, R. T. and Wagner, G. (1992) A triple-resonance pulse scheme for selectively correlating amide 1HN and 15N nuclei with the 1Hα proton of the preceding residue. J. Biomol. NMR 2, 389–394.PubMedCrossRefGoogle Scholar
  31. 31.
    Clubb, R. T., Thanabal, V., and Wagner, G. (1992) A constant-time three-dimensional pulse scheme to correlate intraresidue 1HN, 15N and 13C′ chemical shifts in 15N–13C-labelled proteins. J. Magn. Reson. 97, 213–217.Google Scholar
  32. 32.
    Grzesiek, S. and Bax, A. (1992) Correlating backbone amide and side chain resonances in larger proteins by multiple relayed triple resonance experiments. J. Am. Chem. Soc. 114, 6291–6293.CrossRefGoogle Scholar
  33. 33.
    Grzesiek, S. and Bax, A. (1992) An efficient experiment for sequential backbone assignment of medium-sized isotopically enriched proteins. J. Magn. Reson. 99, 201–207.Google Scholar
  34. 34.
    Wittekind, M. and Mueller, L. (1993) HNCACB, a high sensitivity 3D NMR experiment to correlate amide-proton and nitrogen resonances with the alpha-and beta-carbon resonances in proteins. J. Magn. Reson. B 101, 201–205.CrossRefGoogle Scholar
  35. 35.
    Grzesiek, S. and Bax, A. (1993) Amino acid type determination in the sequential assignment procedure of uniformly 13C/15N-enriched proteins. J. Biomol. NMR 3, 185–204.PubMedGoogle Scholar
  36. 36.
    Wang, A. C., Lodi, P. J., Qin, J., Vulster, G. W., Gronenbom, A. M., and Clore, G. M. (1994) An efficient triple-resonance experiment for proton-directed sequential backbone assignment of medium-sized proteins. J. Magn. Reson. B 105, 196–198.PubMedCrossRefGoogle Scholar
  37. 37.
    Weisemann, R., Ruterjans, H., and 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, 113–120.PubMedCrossRefGoogle Scholar
  38. 38.
    Seip, S., Balbach, J., and Kessler, H. (1993) A simple way for sequential assignment in isotopically enriched proteins using a H(N)CACO correlation. J. Biomol. NMR 3, 233–237.CrossRefGoogle Scholar
  39. 39.
    Szyperski, T., Wider, G., Bushweller, J. H., and Wuthrich, K. (1993) 3D 13C-15N-heteronuclear two-spin coherence spectroscopy for polypeptide backbone assignments in 13C-15N-double-labelled proteins. J. Biomol. NMR 3, 127–132.PubMedGoogle Scholar
  40. 40.
    Bax, A., Clore, G. M., Driscoll, P. C., Gronenborn, A. M., Ikura, M., and Kay, L. E. (1990) Practical aspects of proton-carbon-carbon-proton three-dimensional correlation spectroscopy of 13C-labelled proteins. J. Magn. Reson. 87, 620–627.Google Scholar
  41. 41.
    Bax, A., Clore, G. M., and Gronenborn, A. M. (1990) 1H–1H correlation via isotropic mixing of 13C magnetization, a new three-dimensional approach for assigning 1H and 13C spectra of 13C-enriched proteins. J. Magn. Reson. 88, 425–431.Google Scholar
  42. 42.
    Weisemann, R., Lohr, F., and Ruterjans, H. (1994) HNCCH-TOCSY, a triple resonance experiment for the correlation of backbone 13Cα and 15N resonances with aliphatic side-chain proton resonances and for measuring vicinal 13CO, 1Hβ coupling constants in proteins. J. Biomol. NMR 4, 587–593.PubMedCrossRefGoogle Scholar
  43. 43.
    Grzesiek, S., Anglister, J., and 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, 114–119.CrossRefGoogle Scholar
  44. 44.
    Anglister, J., Grzesiek, S., Wang, A., Ren, H., Klee, C. B., and Bax, A. (1994) 1H, 13C, 15N nuclear magnetic resonance backbone assignments and secondary structure of human calcineurin B. Biochemistry 33, 3540–3547.PubMedCrossRefGoogle Scholar
  45. 45.
    Campbell-Burk, S. L., Domaille, P. J., Starovasnik, M. A., Boucher, W., and Laue, E. D. (1992) Sequential assignment of the backbone nuclei (1H, 15N and r13C) of c-H-ras p21 (1–166). GDP using a novel 4D NMR strategy. J. Biomol. NMR 2, 639–646.PubMedCrossRefGoogle Scholar
  46. 46.
    Boucher, W., Laue, E. D., Campbell-Burk, S. L., and Domaille, P. J. (1992) Four-dimensional heteronuclear triple resonance NMR methods for the assignment of backbone nuclei in proteins. J. Am. Chem. Soc. 114, 2262–2264.CrossRefGoogle Scholar
  47. 47.
    Boucher, W., Laue, E. D., Campbell-Burk, S. L., and Domaille, P. J. (1992) Improved 4D NMR experiments for the assignment of backbone nuclei in 13C/15N labelled proteins. J. Biomol. NMR 2, 631–637.CrossRefGoogle Scholar
  48. 48.
    Kay, L. E., Wittekind, M., McCoy, M. A., Friedrichs, M. S., and Mueller, L. (1992) 4D NMR triple-resonance experiments for assignment of protein back-bone nuclei using shared constant-time evolution periods. J. Magn. Reson. 98, 443–450.Google Scholar
  49. 49.
    Olejniczak, E. T., Xu, R. X., Petros, A. M., and Fesik, S. W. (1992) Optimized constant-time 4D HNCAHA and HN(CO)CAHA experiments. Applications to the backbone assignments of the FKBP/ascomycin complex. J. Magn. Reson. 100, 444–450.Google Scholar
  50. 50.
    Clowes, R. T., Boucher, W., Hardman, C. H., Domaille, P. J., and Laue, E. D. (1993) A 4D HCC(CO)NNH experiment for the correlation of ahphatic side-chain and backbone resonances in 13C/15N-labelled proteins. J. Biomol. NMR 3, 349–354.CrossRefGoogle Scholar
  51. 51.
    Remerowski, M. L., Domke, T., Groenewegen, A., Pepermans, H. A. M., Hilbers, C. W., and van der Ven, F. J. M. (1994) 1H, 13C and 15N NMR backbone assignments and secondary structure of the 269-residue protease subtilism 309 from Bacillus lentus. J. Biomol. NMR 4, 257–278.PubMedCrossRefGoogle Scholar
  52. 52.
    Grzesiek, S., Anglister, J., Ren, H., and Bax, A. (1993) 13C line narrowing by 2H decoupling in 2H/13C/15N-enriched proteins. Application to triple resonance 4D J connectivity of sequential amides. J. Am. Chem. Soc. 115, 4369,4370.CrossRefGoogle Scholar
  53. 53.
    Kraulis, P. J., Domaille, P. J., Campbell-Burk, S. L., Aken, T. V., and Laue, E. D. (1994) Solution structure and dynamics of Ras p21 GDP determined by heteronuclear three-and four-dimensional NMR spectroscopy. Biochemistry 33, 3515–3531.PubMedCrossRefGoogle Scholar
  54. 54.
    Pachter, R., Arrowsmith, C. H., and Jardetzky, O. (1992) The effect of selective deuteration on magnetization transfer in larger proteins. J. Biomol. NMR 2, 183–194.PubMedCrossRefGoogle Scholar
  55. 55.
    LeMaster, D. M. (1994) Isotope labelling in solution protein assignment and structural analysis. Prog. NMR Spectrosc. 26, 371–419.CrossRefGoogle Scholar
  56. 56.
    Wagner, G. (1993) Prospects for NMR of large proteins. J. Biomol. NMR 3, 375–385.PubMedCrossRefGoogle Scholar
  57. 57.
    Sorensen, O. W. (1990) Aspects and prospects of multidimensional time-domain spectroscopy. J. Magn. Reson. 89, 210–216.Google Scholar
  58. 58.
    Farmer, B. T. (1991) Simultaneous [13C, 15N]-HMQC, a pseudo-triple-resonance experiment. J. Magn. Reson. 93, 635–641.Google Scholar
  59. 59.
    Pascal, S. M., Muhandiram, D. R., Yamazaki, T., Forman-Kay, J. D., and Kay, L. E. (1994) Simultaneous acquisition of 13N-and 13C-edited NOE spectra of proteins dissolved in H2O. J. Magn. Reson. Series B 103, 197–201.CrossRefGoogle Scholar
  60. 60.
    Mariam, M., Tessari, M., Boelens, R., Vis, H., and Kaptein, R. (1994) Assignment of the protein backbone from a single 3D, 15N, 13C, time-shared HXYH experiment. J. Magn. Reson. Series B 104, 294–297.CrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 1997

Authors and Affiliations

  • Brian Whitehead
    • 1
  • C. Jeremy Craven
    • 1
  • Jonathan P. Waltho
    • 1
  1. 1.Department of Molecular Biology and BiotechnologyUniversity of SheffieldUK

Personalised recommendations