15N transverse relaxation measurements for the characterization of µs–ms dynamics are deteriorated by the deuterium isotope effect on 15N resulting from solvent exchange

  • Pratibha Kumari
  • Lukas Frey
  • Alexander Sobol
  • Nils-Alexander Lakomek
  • Roland Riek


15N R2 relaxation measurements are key for the elucidation of the dynamics of both folded and intrinsically disordered proteins (IDPs). Here we show, on the example of the intrinsically disordered protein α-synuclein and the folded domain PDZ2, that at physiological pH and near physiological temperatures amide—water exchange can severely skew Hahn-echo based 15N R2 relaxation measurements as well as low frequency data points in CPMG relaxation dispersion experiments. The nature thereof is the solvent exchange with deuterium in the sample buffer, which modulates the 15N chemical shift tensor via the deuterium isotope effect, adding to the apparent relaxation decay which leads to systematic errors in the relaxation data. This results in an artificial increase of the measured apparent 15N R2 rate constants—which should not be mistaken with protein inherent chemical exchange contributions, Rex, to 15N R2. For measurements of 15N R2 rate constants of IDPs and folded proteins at physiological temperatures and pH, we recommend therefore the use of a very low D2O molar fraction in the sample buffer, as low as 1%, or the use of an external D2O reference along with a modified 15N R2 Hahn-echo based experiment. This combination allows for the measurement of Rex contributions to 15N R2 originating from conformational exchange in a time window from µs to ms.


Intrinsically disordered proteins NMR relaxation experiments Amide exchange Deuterium isotope effect Loop dynamics 

Supplementary material

10858_2018_211_MOESM1_ESM.pdf (409 kb)
Supplementary material 1 (PDF 409 KB)


  1. Abyzov A et al (2016) Identification of dynamic modes in an intrinsically disordered protein using temperature-dependent NMR relaxation. J Am Chem Soc 138:6240–6251CrossRefGoogle Scholar
  2. Arai M, Sugase K, Dyson HJ, Wright PE (2015) Conformational propensities of intrinsically disordered proteins influence the mechanism of binding and folding. Proc Natl Acad Sci USA 112:9614–9619ADSCrossRefGoogle Scholar
  3. Bah A et al (2015) Folding of an intrinsically disordered protein by phosphorylation as a regulatory switch. Nature 519:106–240ADSCrossRefGoogle Scholar
  4. Bai YW, Milne JS, Mayne L, Englander SW (1993) Primary structure effects on peptide group hydrogen-exchange. Protein Struct Funct Genet 17:75–86CrossRefGoogle Scholar
  5. Bouvignies G et al (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
  6. Camilloni C, Vendruscolo M (2012) NMR chemical shifts and protein dynamics. FEBS J 279:529–529Google Scholar
  7. Campioni S et al (2014) The presence of an air-water interface affects formation and elongation of alpha-Synuclein fibrils. J Am Chem Soc 136:2866–2875CrossRefGoogle Scholar
  8. Case DA (2013) Chemical shifts in biomolecules. Curr Opin Struct Biol 23:172–176CrossRefGoogle Scholar
  9. Cavanagh J, Fairbrother WJ, Palmer AG, Rance M, Skelton NJ (2007) Protein NMR spectroscopy. Academic Press, New YorkGoogle Scholar
  10. Charlier C et al (2013) Nanosecond time scale motions in proteins revealed by high-resolution NMR relaxometry. J Am Chem Soc 135:18665–18672CrossRefGoogle Scholar
  11. Charlier C, Cousin SF, Ferrage F (2016) Protein dynamics from nuclear magnetic relaxation. Chem Soc Rev 45:2410–2422CrossRefGoogle Scholar
  12. Charlier C et al (2017) Structure and dynamics of an intrinsically disordered protein region that partially folds upon binding by chemical-exchange NMR. J Am Chem Soc 139:12219–12227CrossRefGoogle Scholar
  13. Connelly GP, Bai YW, Jeng MF, Englander SW (1993) Isotope effects in peptide group hydrogen-exchange. Protein Struct Funct Genet 17:87–92CrossRefGoogle Scholar
  14. Croke RL, Sallum CO, Watson E, Watt ED, Alexandrescu AT (2008) Hydrogen exchange of monomeric alpha-synuclein shows unfolded structure persists at physiological temperature and is independent of molecular crowding in Escherichia coli. Prot Sci 17:1434–1445CrossRefGoogle Scholar
  15. Delaforge E et al (2018) Deciphering the dynamic interaction profile of an intrinsically disordered protein by NMR exchange spectroscopy. J Am Chem Soc 140:1148–1158CrossRefGoogle Scholar
  16. Dempsey CE (2001) Hydrogen exchange in peptides and proteins using NMR-spectroscopy. Prog Nucl Magn Reson Spectrosc 39:135–170CrossRefGoogle Scholar
  17. Farrow NA et al (1994) Backbone dynamics of a free and a phosphopeptide-complexed Src homology-2 domain studied by N-15 Nmr relaxation. Biochemistry 33:5984–6003CrossRefGoogle Scholar
  18. Favier A, Brutscher B (2011) Recovering lost magnetization: polarization enhancement in biomolecular NMR. J Biomol NMR 49:9–15CrossRefGoogle Scholar
  19. Fawzi NL, Ying JF, Ghirlando R, Torchia DA, Clore GM (2011) Atomic-resolution dynamics on the surface of amyloid-beta protofibrils probed by solution NMR. Nature 480:268–361ADSCrossRefGoogle Scholar
  20. Fenwick RB et al (2011) Weak long-range correlated motions in a surface patch of ubiquitin involved in molecular recognition. J Am Chem Soc 133:10336–10339CrossRefGoogle Scholar
  21. Gianni S et al (2005) The kinetics of PDZ domain-ligand interactions and implications for the binding mechanism. J Biol Chem 280:34805–34812CrossRefGoogle Scholar
  22. Hansel R, Luh LM, Corbeski I, Trantirek L, Dotsch V (2014) In-Cell NMR and EPR Spectroscopy of Biomacromolecules. Angew Chem Int Ed Engl 53:10300–10314CrossRefGoogle Scholar
  23. Henry GD, Weiner JH, Sykes BD (1987) Backbone dynamics of a model membrane-protein—measurement of individual amide hydrogen-exchange rates in detergent-Solubilized M13 coat protein using C-13 Nmr hydrogen-deuterium isotope shifts. Biochemistry 26:3626–3634CrossRefGoogle Scholar
  24. Huang C, Ren G, Zhou H, Wang CC (2005) A new method for purification of recombinant human alpha-synuclein in Escherichia coli. Protein Expr Purif 42:173–177CrossRefGoogle Scholar
  25. Ishima R, Torchia DA (1999) Estimating the time scale of chemical exchange of proteins from measurements of transverse relaxation rates in solution. J Biomol NMR 14:369–372CrossRefGoogle Scholar
  26. Iwahara J, Clore GM (2006) Detecting transient intermediates in macromolecular binding by paramagnetic NMR. Nature 440:1227–1230ADSCrossRefGoogle Scholar
  27. Johnson M, Coulton AT, Geeves MA, Mulvihill DP (2010) Targeted amino-terminal acetylation of recombinant proteins in E. coli. PLoS One 5:e15801ADSCrossRefGoogle Scholar
  28. Kannan A, Camilloni C, Sahakyan AB, Cavalli A, Vendruscolo MA (2014) Conformational ensemble derived using nmr methyl chemical shifts reveals a mechanical clamping transition that gates the binding of the HU protein to DNA. J Am Chem Soc 136:2204–2207CrossRefGoogle Scholar
  29. Kateb F, Pelupessy P, Bodenhausen G (2007) Measuring fast hydrogen exchange rates by NMR spectroscopy. J Magn Reson 184:108–113ADSCrossRefGoogle Scholar
  30. Kay LE, Torchia DA, Bax A (1989) Backbone dynamics of proteins as studied by N-15 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. Khare D, Alexander P, Orban J (1999) Hydrogen bonding and equilibrium protium-deuterium fractionation factors in the immunoglobulin G binding domain of protein G. Biochemistry 38:3918–3925CrossRefGoogle Scholar
  33. Kim S, Wu KP, Baum J (2013a) Fast hydrogen exchange affects (1)(5)N relaxation measurements in intrinsically disordered proteins. J Biomol NMR 55:249–256Google Scholar
  34. Kim S, Wu KP, Baum J (2013b) Fast hydrogen exchange affects N-15 relaxation measurements in intrinsically disordered proteins. J Biomol NMR 55:249–256CrossRefGoogle Scholar
  35. Kiteyski-LeBlanc JL et al (2018) Investigating the dynamics of destabilized nucleosomes using methyl-TROSY NMR. J Am Chem Soc 140:4774–4777CrossRefGoogle Scholar
  36. Kurzbach D, Kontaxis G, Coudevylle N, Konrat R (2015) NMR spectroscopic studies of the conformational ensembles of intrinsically disordered proteins. Intrinsically Disordered Proteins Stud Nmr Spectrosc 870:149–185CrossRefGoogle Scholar
  37. Lakomek NA, Ying JF, Bax A (2012) Measurement of N-15 relaxation rates in perdeuterated proteins by TROSY-based methods. J Biomol NMR 53:209–221CrossRefGoogle Scholar
  38. Lakomek NA et al (2013) Internal dynamics of the homotrimeric HIV-1 viral coat protein gp41 on multiple time scales. Angew Chem Int Ed Engl 52:3911–3915CrossRefGoogle Scholar
  39. Lakomek NA, Draycheva A, Bornemann T, Wintermeyer W (2016) Electrostatics and intrinsic disorder drive translocon binding of the SRP receptor FtsY. Angew Chem Int Ed Engl 55:9544–9547CrossRefGoogle Scholar
  40. Lange OF et al (2008) Recognition dynamics up to microseconds revealed from an RDC-derived ubiquitin ensemble in solution. Science 320:1471–1475ADSCrossRefGoogle Scholar
  41. Lindorff-Larsen K, Best RB, DePristo MA, Dobson CM, Vendruscolo M (2005) Simultaneous determination of protein structure and dynamics. Nature 433:128–132ADSCrossRefGoogle Scholar
  42. Lippens G et al (2018) In-cell NMR: from metabolites to macromolecules. Analyst 143:620–629ADSCrossRefGoogle Scholar
  43. Lopez J, Schneider R, Cantrelle FX, Huvent I, Lippens G (2016) studying intrinsically disordered proteins under true in vivo conditions by combined cross-polarization and carbonyl-detection NMR spectroscopy. Angew Chem Int Ed Engl 55:7418–7422CrossRefGoogle Scholar
  44. Loria JP, Rance M, Palmer AG (1999) A relaxation-compensated Carr-Purcell-Meiboom-Gill sequence for characterizing chemical exchange by NMR spectroscopy. J Am Chem Soc 121:2331–2332CrossRefGoogle Scholar
  45. Luchinat E et al. (2014) In-cell NMR reveals potential precursor of toxic species from SOD1 fALS mutants. Nat Commun 5:5502CrossRefGoogle Scholar
  46. Luginbuhl P, Wuthrich K (2002) Semi-classical nuclear spin relaxation theory revisited for use with biological macromolecules. Prog Nucl Magn Reson Spectrosc 40:199–247CrossRefGoogle Scholar
  47. Maltsev AS, Chen J, Levine RL, Bax A (2013) Site-specific interaction between alpha-synuclein and membranes probed by NMR-observed methionine oxidation rates. J Am Chem Soc 135:2943–2946CrossRefGoogle Scholar
  48. Markwick PRL, Showalter SA, Bouvignies G, Bruschweiler R, Blackledge M (2009) Structural dynamics of protein backbone phi angles: extended molecular dynamics simulations versus experimental (3) J scalar couplings. J Biomol NMR 45:17–21CrossRefGoogle Scholar
  49. Millet O, Loria JP, Kroenke CD, Pons M, Palmer AG (2000) The static magnetic field dependence of chemical exchange linebroadening defines the NMR chemical shift time scale. J Am Chem Soc 122:2867–2877CrossRefGoogle Scholar
  50. Mittermaier A, Kay LE (2006) Review—new tools provide new insights in NMR studies of protein dynamics. Science 312:224–228ADSCrossRefGoogle Scholar
  51. Mittermaier AK, Kay LE (2009) Observing biological dynamics at atomic resolution using NMR. Trends Biochem Sci 34:601–611CrossRefGoogle Scholar
  52. Mulder FAA, Skrynnikov NR, Hon B, Dahlquist FW, Kay LE (2001) Measurement of slow (mu s-ms) time scale dynamics in protein side chains by N-15 relaxation dispersion NMR spectroscopy: application to Asn and Gln residues in a cavity mutant of T4 lysozyme. J Am Chem Soc 123:967–975CrossRefGoogle Scholar
  53. Palmer AG (2004) NMR characterization of the dynamics of biomacromolecules. Chem Rev 104:3623–3640CrossRefGoogle Scholar
  54. Palmer AG (2015) Enzyme dynamics from NMR spectroscopy. Acc Chem Res 48:457–465CrossRefGoogle Scholar
  55. Palmer AG, Kroenke CD, Loria JP (2001) Nuclear magnetic resonance methods for quantifying microsecond-to-millisecond motions in biological macromolecules. Nuclear Magn Reson Biol Macromol Pt B 339:204–238Google Scholar
  56. Pelupessy P, Ravindranathan S, Bodenhausen G (2003) Correlated motions of successive amide N-H bonds in proteins. J Biomol NMR 25:265–280CrossRefGoogle Scholar
  57. Pervushin K, Riek R, Wider G, Wuthrich K, 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, 12366–12371 (1997)ADSCrossRefGoogle Scholar
  58. Pervushin KV, Wider G, Wuthrich K (1998) Single transition-to-single transition polarization transfer (ST2-PT) in [N-15,H-1]-TROSY. J Biomol NMR 12:345–348CrossRefGoogle Scholar
  59. 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
  60. Pintacuda G, Otting G (2002) Identification of protein surfaces by NMR measurements with a paramagnetic Gd(III) chelate. J Am Chem Soc 124:372–373CrossRefGoogle Scholar
  61. Plitzko JM, Schuler B, Selenko P (2017) Structural biology outside the box—inside the cell. Curr Opin Struct Biol 46:110–121CrossRefGoogle Scholar
  62. Reckel S, Hansel R, Lohr F, Dotsch V (2007) In-cell NMR spectroscopy. Prog Nucl Magn Reson Spectrosc 51:91–101CrossRefGoogle Scholar
  63. Rezaei-Ghaleh N, Blackledge M, Zweckstetter M (2012) Intrinsically disordered proteins: from sequence and conformational properties toward drug discovery. Chembiochem 13:930–950CrossRefGoogle Scholar
  64. Salvi N, Ulzega S, Ferrage F, Bodenhausen G (2012) Time scales of slow motions in ubiquitin explored by heteronuclear double resonance. J Am Chem Soc 134:2481–2484CrossRefGoogle Scholar
  65. Salvi N, Abyzov A, Blackledge M (2017) Atomic resolution conformational dynamics of intrinsically disordered proteins from NMR spin relaxation. Prog Nucl Magn Reson Spectrosc 102:43–60CrossRefGoogle Scholar
  66. Schneider R et al (2015) Visualizing the molecular recognition trajectory of an intrinsically disordered protein using multinuclear relaxation dispersion. NMR J Am Chem Soc 137:1220–1229CrossRefGoogle Scholar
  67. 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
  68. Skrynnikov NR, Ernst RR (1999) Detection of intermolecular chemical exchange through decorrelation of two-spin order. J Magn Reson 137:276–280ADSCrossRefGoogle Scholar
  69. Sugase K, Dyson HJ, Wright PE (2007) Mechanism of coupled folding and binding of an intrinsically disordered protein. Nature 447:1021–1025ADSCrossRefGoogle Scholar
  70. Theillet FX et al (2016) Structural disorder of monomeric alpha-synuclein persists in mammalian cells. Nature 530:45–50ADSCrossRefGoogle Scholar
  71. Tolman JR, Al-Hashimi HM, Kay LE, Prestegard JH (2001) Structural and dynamic analysis of residual dipolar coupling data for proteins. J Am Chem Soc 123:1416–1424CrossRefGoogle Scholar
  72. Tompa P (2002) Intrinsically unstructured proteins. Trends Biochem Sci 27:527–533CrossRefGoogle Scholar
  73. Tugarinov V (2014) Indirect use of deuterium in solution NMR studies of protein structure and hydrogen bonding. Prog Nucl Magn Reson Spectrosc 77:49–68CrossRefGoogle Scholar
  74. Tugarinov V, Kay LE (2005) Methyl groups as probes of structure and dynamics in NMR studies of high-molecular-weight proteins. Chembiochem 6:1567–1577CrossRefGoogle Scholar
  75. Vallurupalli P, Bouvignies G, Kay LE (2012) Studying “Invisible” excited protein states in slow exchange with a major state conformation. J Am Chem Soc 134:8148–8161CrossRefGoogle Scholar
  76. Vasos PR, Hall JB, Kummerle R, Fushman D (2006) Measurement of N-15 relaxation in deuterated amide groups in proteins using direct nitrogen detection. J Biomol NMR 36:27–36CrossRefGoogle Scholar
  77. Vogeli B (2017) Cross-correlated relaxation rates between protein backbone H-X dipolar interactions. J Biomol NMR 67:211–232CrossRefGoogle Scholar
  78. Vogeli B, Yao LS (2009) Correlated dynamics between protein HN and HC bonds observed by NMR cross relaxation. J Am Chem Soc 131:3668–3678CrossRefGoogle Scholar
  79. Vogeli B, Kazemi S, Guntert P, Riek R (2012) Spatial elucidation of motion in proteins by ensemble-based structure calculation using exact NOEs. Nat Struct Mol Biol 19:1053–1110CrossRefGoogle Scholar
  80. Vogeli B et al (2014) Towards a true protein movie: a perspective on the potential impact of the ensemble-based structure determination using exact NOEs. J Magn Reson 241:53–59ADSCrossRefGoogle Scholar
  81. Wagner G, Wuthrich K (1979) Structural interpretation of the amide proton-exchange in the basic pancreatic trypsin-inhibitor and related proteins. J Mol Biol 134:75–94CrossRefGoogle Scholar
  82. Wang C, Rance M, Palmer AG 3rd (2003) Mapping chemical exchange in proteins with MW> 50 kD. J Am Chem Soc 125:8968–8969CrossRefGoogle Scholar
  83. Wright PE, Dyson HJ (1999) Intrinsically unstructured proteins: re-assessing the protein structure-function paradigm. J Mol Biol 293:321–331CrossRefGoogle Scholar
  84. Wright PE, Dyson HJ (2015) Intrinsically disordered proteins in cellular signalling and regulation. Nat Rev Mol Cell Biol 16:18–29CrossRefGoogle Scholar
  85. Xu J, Millet O, Kay LE, Skrynnikov NR (2005) New spin probe of protein dynamics: Nitrogen relaxation in N-15-H-2 amide groups. J Am Chem Soc 127:3220–3229CrossRefGoogle Scholar
  86. Xu XF et al (2008) Dynamics in a pure encounter complex of two proteins studied by solution scattering and paramagnetic NMR spectroscopy. J Am Chem Soc 130:6395–6403CrossRefGoogle Scholar
  87. Yuwen T, Skrynnikov NR (2014) CP-HISQC: a better version of HSQC experiment for intrinsically disordered proteins under physiological conditions. J Biomol NMR 58:175–192CrossRefGoogle Scholar
  88. Yuwen T, Skrynnikov NR, Proton-decoupled CPMG (2014) A better experiment for measuring N-15 R-2 relaxation in disordered proteins. J Magn Reson 241:155–169ADSCrossRefGoogle Scholar
  89. Zweckstetter M (2016) Intrinsically disordered proteins in neurodegeneration Markus Zweckstetter. Biophys J 110:2aCrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2018

Authors and Affiliations

  1. 1.Laboratory of Physical Chemistry, Department of Chemistry and Applied BiosciencesETH ZürichZurichSwitzerland

Personalised recommendations