Journal of Biomolecular NMR

, Volume 53, Issue 3, pp 209–221 | Cite as

Measurement of 15N relaxation rates in perdeuterated proteins by TROSY-based methods

  • Nils-Alexander Lakomek
  • Jinfa Ying
  • Ad Bax


While extracting dynamics parameters from backbone 15N relaxation measurements in proteins has become routine over the past two decades, it is increasingly recognized that accurate quantitative analysis can remain limited by the potential presence of systematic errors associated with the measurement of 15N R1 and R2 or R relaxation rates as well as heteronuclear 15N-{1H} NOE values. We show that systematic errors in such measurements can be far larger than the statistical error derived from either the observed signal-to-noise ratio, or from the reproducibility of the measurement. Unless special precautions are taken, the problem of systematic errors is shown to be particularly acute in perdeuterated systems, and even more so when TROSY instead of HSQC elements are used to read out the 15N magnetization through the NMR-sensitive 1H nucleus. A discussion of the most common sources of systematic errors is presented, as well as TROSY-based pulse schemes that appear free of systematic errors to the level of <1 %. Application to the small perdeuterated protein GB3, which yields exceptionally high S/N and therefore is an ideal test molecule for detection of systematic errors, yields relaxation rates that show considerably less residue by residue variation than previous measurements. Measured R2′/R1′ ratios fit an axially symmetric diffusion tensor with a Pearson’s correlation coefficient of 0.97, comparable to fits obtained for backbone amide RDCs to the Saupe matrix.


Backbone dynamics Relaxation TROSY Perdeuterated proteins Cross-correlated relaxation Water saturation 



We thank Dennis A Torchia and Alex Grishaev for helpful discussions and computer simulations, Frank Delaglio for assistance with the evaluation of relaxation data and Alex Maltsev for help with the expression and purification of GB3. This work was funded by the Intramural Research Program of the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health (NIH) and the Intramural AIDS-Targeted Antiviral Program of the Office of the Director, NIH.

Supplementary material

10858_2012_9626_MOESM1_ESM.pdf (574 kb)
Supplementary material 1 (PDF 573 kb)


  1. Bothnerby AA, Shukla R (1988) Spin-locked states of homonuclear 2-spin systems. J Magn Reson 77:524–535Google Scholar
  2. Boyd J, Hommel U, Campbell ID (1990) Influence of cross-correlation between dipolar and anisotropic chemical shift relaxation mechanisms upon longitudinal relaxation rates of 15 N in macromolecules. Chem Phys Lett 175:477–482ADSCrossRefGoogle Scholar
  3. Burum DP, Ernst RR (1980) Net polarization transfer via a J-ordered state for signal enhancement of low-sensitivity nuclei. J Magn Reson 39:163–168Google Scholar
  4. Cavanagh J, Fairbrother WJ, Palmer AG, Rance M, Skelton N (2007) Protein NMR spectroscopy: principles and practice. Elsevier Academic Press, BurlingtonGoogle Scholar
  5. Chen K, Tjandra N (2011) Water proton spin saturation affects measured protein backbone (15)N spin relaxation rates. J Magn Reson 213:151–157ADSCrossRefGoogle Scholar
  6. Chiarparin E, Pelupessy P, Ghose R, Bodenhausen G (1999) Relaxation of two-spin coherence due to cross-correlated fluctuations of dipole–dipole couplings and anisotropic shifts in NMR of N-15,C-13-labeled biomolecules. J Am Chem Soc 121:6876–6883CrossRefGoogle Scholar
  7. Chill JH, Louis JM, Baber JL, Bax A (2006) Measurement of N-15 relaxation in the detergent-solubilized tetrameric KcsA potassium channel. J Biomol NMR 36:123–136CrossRefGoogle Scholar
  8. 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
  9. Farrow NA, Muhandiram R, Singer AU, Pascal SM, Kay CM, Gish G, Shoelson SE, Pawson T, Forman-Kay JD, Kay LE (1994) Backbone dynamics of a free and a phosphopeptide-complexed Src homology 2 domain studied by 15N NMR relaxation. Biochemistry 33:5984–6003CrossRefGoogle Scholar
  10. Favier A, Brutscher B (2011) Recovering lost magnetization: polarization enhancement in biomolecular NMR. J Biomol NMR 49:9–15CrossRefGoogle Scholar
  11. Felli IC, Desvaux H, Bodenhausen G (1998) Local mobility of N-15 labeled biomolecules characterized through cross-correlation rates: applications to paramagnetic proteins. J Biomol NMR 12:509–521CrossRefGoogle Scholar
  12. Ferrage F, Piserchio A, Cowburn D, Ghose R (2008) On the measurement of N-15-{H-1} nuclear Overhauser effects. J Magn Reson 192:302–313ADSCrossRefGoogle Scholar
  13. Ferrage F, Cowburn D, Ghose R (2009) Accurate sampling of high-frequency motions in proteins by steady-state (15)N-{(1)H} nuclear Overhauser effect measurements in the presence of cross-correlated relaxation. J Am Chem Soc 131: 6048–+Google Scholar
  14. Ferrage F, Reichel A, Battacharya S, Cowburn D, Ghose R (2010) On the measurement of (15)N-{(1)H} nuclear Overhauser effects. 2. Effects of the saturation scheme and water signal suppression. J Magn Reson 207:294–303ADSCrossRefGoogle Scholar
  15. Freeman R, Kempsell SP, Levitt MH (1980) Radiofrequency pulse sequences which compensate their own imperfections. J Magn Reson 38:453–479Google Scholar
  16. Fushman D, Cowburn D (1998) Model-independent analysis of N-15 chemical shift anisotropy from NMR relaxation data. Ubiquitin as a test example. J Am Chem Soc 120:7109–7110CrossRefGoogle Scholar
  17. Fushman D, Cowburn D (1999) The effect of noncollinearity of N-15-H-1 dipolar and N-15 CSA tensors and rotational anisotropy on N-15 relaxation, CSA/dipolar cross correlation, and TROSY. J Biomol NMR 13:139–147CrossRefGoogle Scholar
  18. Fushman D, Tjandra N, Cowburn D (1998) Direct measurement of N-15 chemical shift anisotropy in solution. J Am Chem Soc 120:10947–10952CrossRefGoogle Scholar
  19. Fushman D, Xu R, Cowburn D (1999) Direct determination of changes of interdomain orientation on ligation: use of the orientational dependence of N-15 NMR relaxation in Abl SH(32). Biochemistry 38:10225–10230CrossRefGoogle Scholar
  20. Garwood M, Ke Y (1991) Symmetrical pulses to induce arbitrary flip angles with compensation for RF inhomogeneity and resonance offsets. J Magn Reson 94:511–525Google Scholar
  21. Geen H, Freeman R (1991) Band-selective radiofrequency pulses. J Magn Reson 93:93–141Google Scholar
  22. Grzesiek S, Bax A (1993) The importance of not saturating H2O in protein NMR. Application to sensitivity enhancement and NOE measurement. J Am Chem Soc 115:12593CrossRefGoogle Scholar
  23. Hall JB, Fushman D (2003) Characterization of the overall and local dynamics of a protein with intermediate rotational anisotropy: differentiating between conformational exchange and anisotropic diffusion in the B3 domain of protein G. J Biomol NMR 27:261–275CrossRefGoogle Scholar
  24. Hall JB, Fushman D (2006) Variability of the N-15 chemical shielding tensors in the B3 domain of protein G from N-15 relaxation measurements at several fields. Implications for backbone order parameters. J Am Chem Soc 128:7855–7870CrossRefGoogle Scholar
  25. Hansen DF, Kay LE (2007) Improved magnetization alignment schemes for spin-lock relaxation experiments. J Biomol NMR 37:245–255CrossRefGoogle Scholar
  26. Hare BJ, Wyss DF, Osburne MS, Kern PS, Reinherz EL, Wagner G (1999) Structure, specificity and CDR mobility of a class II restricted single-chain T-cell receptor. Nat Struct Biol 6:574–581CrossRefGoogle Scholar
  27. Hornak V, Abel R, Okur A, Strockbine B, Roitberg A, Simmerling C (2006) Comparison of multiple amber force fields and development of improved protein backbone parameters. Prot Struct Funct Bioinform 65:712–725CrossRefGoogle Scholar
  28. Ishima R, Torchia DA (2000) Protein dynamics from NMR. Nat Struct Biol 7:740–743CrossRefGoogle Scholar
  29. Kay LE (1998) Protein dynamics from NMR. Nat Struct Biol 513–517Google Scholar
  30. Kay LE, Torchia DA, Bax A (1989) Backbone dynamics of proteins as studied by 15 N inverse detected heteronuclear NMR spectroscopy: application to staphylococcal nuclease. Biochemistry 28:8972–8979CrossRefGoogle Scholar
  31. 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
  32. Kern D, Zuiderweg ERP (2003) The role of dynamics in allosteric regulation. Curr Opin Struct Biol 13:748–757CrossRefGoogle Scholar
  33. Kobzar K, Skinner TE, Khaneja N, Glaser SJ, Luy B (2008) Exploring the limits of broadband excitation and inversion: II. Rf-power optimized pulses. J Magn Reson 194:58–66ADSCrossRefGoogle Scholar
  34. Korzhnev DM, Skrynnikov NR, Millet O, Torchia DA, Kay LE (2002) An NMR experiment for the accurate measurement of heteronuclear spin-lock relaxation rates. J Am Chem Soc 124:10743–10753CrossRefGoogle Scholar
  35. Lee AL, Wand AJ (2001) Microscopic origins of entropy, heat capacity and the glass transition in proteins. Nature 411:501–504ADSCrossRefGoogle Scholar
  36. Lee AL, Kinnear SA, Wand AJ (2000) Redistribution and loss of side chain entropy upon formation of a calmodulin-peptide complex. Nat Struct Biol 7:72–77CrossRefGoogle Scholar
  37. Li D-W, Brueschweiler R (2009) A dictionary for protein side-chain entropies from NMR order parameters. J Am Chem Soc 131:7226–7227CrossRefGoogle Scholar
  38. Markley JL, Horsley WJ, Klein MP (1971) Spin-lattice relaxation measurements in slowly relaxing complex spectra. J Chem Phys 55:3604–3605Google Scholar
  39. Massi F, Johnson E, Wang CY, Rance M, Palmer AG (2004) NMR R-1 rho rotating-frame relaxation with weak radio frequency fields. J Am Chem Soc 126:2247–2256CrossRefGoogle Scholar
  40. Millet O, Muhandiram DR, Skrynnikov NR, Kay LE (2002) Deuterium spin probes of side-chain dynamics in proteins. 1. Measurement of five relaxation rates per deuteron in C-13-labeled and fractionally H-2-enriched proteins in solution. J Am Chem Soc 124:6439–6448CrossRefGoogle Scholar
  41. Mitschang L, Keeler J, Davis AL, Oschkinat H (1992) Removal of zero-quantum interference in NOESY spectra of proteins by utilizing the natural inhomogeneity of the radiofrequency field. J Biomol NMR 2:545–556CrossRefGoogle Scholar
  42. Mittag T, Kay LE, Forman-Kay JD (2010) Protein dynamics and conformational disorder in molecular recognition. J Mol Recognit 23:105–116Google Scholar
  43. Mittermaier A, Kay LE (2006) Review—new tools provide new insights in NMR studies of protein dynamics. Science 312:224–228ADSCrossRefGoogle Scholar
  44. Muhandiram DR, Yamaaki T, Sykes BD, Kay LE (1995) Measurement of 2H T1 and T1p relaxation times in uniformly 13C-labeled and fractionally 2H-labeled proteins in solution. J Am Chem Soc 117:11536–11544CrossRefGoogle Scholar
  45. Mulder FAA, de Graaf RA, Kaptein R, Boelens R (1998) An off-resonance rotating frame relaxation experiment for the investigation of macromolecular dynamics using adiabatic rotations. J Magn Reson 131:351–357ADSCrossRefGoogle Scholar
  46. Nadaud PS, Helmus JJ, Jaroniec CP (2007) 13C and 15 N chemical shift assignments and secondary structure of the B3 immunoglobulin-binding domain of streptococcal protein G by magic-angle spinning solid-state NMR spectroscopy. Biomol NMR Assign 1:117–120CrossRefGoogle Scholar
  47. Namanja AT, Wang XJ, Xu B, Mercedes-Camacho AY, Wilson KA, Etzkorn FA, Peng JW (2011) Stereospecific gating of functional motions in Pin1. Proc Natl Acad Sci USA 108:12289–12294CrossRefGoogle Scholar
  48. Nietlispach D (2005) Suppression of anti-TROSY lines in a sensitivity enhanced gradient selection TROSY scheme. J Biomol NMR 31:161–166CrossRefGoogle Scholar
  49. Nirmala NR, Wagner G (1988) Measurement Of C-13 relaxation-times in proteins by two-dimensional heteronuclear H1-C-13 correlation spectroscopy. J Am Chem Soc 110:7557–7558CrossRefGoogle Scholar
  50. Palmer AG (2004) NMR characterization of the dynamics of biomacromolecules. Chem Rev (Washington, DC, US) 104:3623–3640Google Scholar
  51. Pelupessy P, Chiarparin E, Ghose R, Bodenhausen G (1999) Simultaneous determination of Psi and Phi angles in proteins from measurements of cross-correlated relaxation effects. J Biomol NMR 14:277–280CrossRefGoogle Scholar
  52. Peng JW, Wagner G (1992) Mapping of spectral density functions using heteronuclear NMR relaxation measurements. J Magn Reson 98:308–332Google Scholar
  53. Peng JW, Thanabal V, Wagner G (1991) 2d heteronuclear NMR measurements of spin-lattice relaxation-times in the rotating frame of X nuclei in heteronuclear HX spin systems. J Magn Reson 94:82–100Google Scholar
  54. 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
  55. Pervushin KV, Wider G, Wuthrich K (1998) Single transition-to-single transition polarization transfer (ST2-PT) in [N15, H1]-TROSY. J Biomol NMR 12:345–348CrossRefGoogle Scholar
  56. Piotto M, Saudek V, Sklenár V (1992) Gradient-tailored excitation for single-quantum NMR spectroscopy of aqueous solutions. J Biomol NMR 2:661–665CrossRefGoogle Scholar
  57. Popovych N, Sun S, Ebright RH, Kalodimos CG (2006) Dynamically driven protein allostery. Nat Struct Mol Biol 13:831–838CrossRefGoogle Scholar
  58. Price DJ, Brooks CL (2002) Modern protein force fields behave comparably in molecular dynamics simulations. J Comput Chem 23:1045–1057CrossRefGoogle Scholar
  59. Reif B, Hennig M, Griesinger C (1997) Direct measurement of angles between bond vectors in high-resolution NMR. Science 276:1230–1233CrossRefGoogle Scholar
  60. Rinnenthal J, Buck J, Ferner J, Wacker A, Fuertig B, Schwalbe H (2011) Mapping the landscape of RNA dynamics with NMR spectroscopy. Acc Chem Res 44:1292–1301CrossRefGoogle Scholar
  61. Showalter SA, Bruschweiler R (2007) Validation of molecular dynamics simulations of biomolecules using NMR spin relaxation as benchmarks: application to the AMBER99SB force field. J Chem Theory Comput 3:961–975CrossRefGoogle Scholar
  62. Sklenar V, Torchia D, Bax A (1987) Measurement of C-13 longitudinal relaxation using H-1 detection. J Magn Reson 73:375–379Google Scholar
  63. Smith MA, Hu H, Shaka AJ (2001) Improved broadband inversion performance for NMR in liquids. J Magn Reson 151:269–283ADSCrossRefGoogle Scholar
  64. Stocker U, van Gunsteren WF (2000) Molecular dynamics simulation of hen egg white lysozyme: a test of the GROMOS96 force field against nuclear magnetic resonance data. Prot Struct Funct Genet 40:145–153CrossRefGoogle Scholar
  65. Stone MJ (2001) NMR relaxation studies of the role of conformational entropy in protein stability and ligand binding. Acc Chem Res 34:379–388CrossRefGoogle Scholar
  66. Tjandra N, Feller SE, Pastor RW, Bax A (1995) Rotational diffusion anisotropy of human ubiquitin from N-15 NMR relaxation. J Am Chem Soc 117:12562–12566CrossRefGoogle Scholar
  67. Tjandra N, Szabo A, Bax A (1996) Protein backbone dynamics and N-15 chemical shift anisotropy from quantitative measurement of relaxation interference effects. J Am Chem Soc 118:6986–6991CrossRefGoogle Scholar
  68. Ulmer TS, Campbell ID, Boyd J (2004) Amide proton relaxation measurements employing a highly deuterated protein. J Magn Reson 166:190–201ADSCrossRefGoogle Scholar
  69. Varadan R, Assfalg M, Haririnia A, Raasi S, Pickart C, Fushman D (2004) Solution conformation of Lys(63)-linked di-ubiquitin chain provides clues to functional diversity of polyubiquitin signaling. J Biol Chem 279:7055–7063CrossRefGoogle Scholar
  70. Walker O, Varadan R, Fushman D (2004) Efficient and accurate determination of the overall rotational diffusion tensor of a molecule from N-15 relaxation data using computer program ROTDIF. J Magn Reson 168:336–345ADSCrossRefGoogle Scholar
  71. Wang AC, Bax A (1993) Minimizing the effects of radiofrequency heating in multidimensional NMR experiments. J Biomol NMR 3:715–720CrossRefGoogle Scholar
  72. Yang DW, Mok YK, FormanKay JD, Farrow NA, Kay LE (1997) Contributions to protein entropy and heat capacity from bond vector motions measured by NMR spin relaxation. J Mol Biol 272:790–804CrossRefGoogle Scholar
  73. Yang DW, Mittermaier A, Mok YK, Kay LE (1998) A study of protein side-chain dynamics from new H-2 auto- correlation and C-13 cross-correlation NMR experiments: application to the N-terminal SH3 domain from drk. J Mol Biol 276:939–954CrossRefGoogle Scholar
  74. Zhu G, Xia YL, Nicholson LK, Sze KH (2000) Protein dynamics measurements by TROSY-based NMR experiments. J Magn Reson 143:423–426ADSCrossRefGoogle Scholar
  75. Zidek L, Novotny MV, Stone MJ (1999) Increased protein backbone conformational entropy upon hydrophobic ligand binding. Nat Struct Biol 6:1118–1121CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. (outside the USA) 2012

Authors and Affiliations

  1. 1.Laboratory of Chemical Physics (LCP), DHHS NIDDK National Institutes of HealthBethesdaUSA

Personalised recommendations