Journal of Biomolecular NMR

, Volume 61, Issue 2, pp 123–136 | Cite as

A minor conformation of a lanthanide tag on adenylate kinase characterized by paramagnetic relaxation dispersion NMR spectroscopy

  • Mathias A. S. Hass
  • Wei-Min Liu
  • Roman V. Agafonov
  • Renee Otten
  • Lien A. Phung
  • Jesika T. Schilder
  • Dorothee Kern
  • Marcellus UbbinkEmail author


NMR relaxation dispersion techniques provide a powerful method to study protein dynamics by characterizing lowly populated conformations that are in dynamic exchange with the major state. Paramagnetic NMR is a versatile tool for investigating the structures and dynamics of proteins. These two techniques were combined here to measure accurate and precise pseudocontact shifts of a lowly populated conformation. This method delivers valuable long-range structural restraints for higher energy conformations of macromolecules in solution. Another advantage of combining pseudocontact shifts with relaxation dispersion is the increase in the amplitude of dispersion profiles. Lowly populated states are often involved in functional processes, such as enzyme catalysis, signaling, and protein/protein interactions. The presented results also unveil a critical problem with the lanthanide tag used to generate paramagnetic relaxation dispersion effects in proteins, namely that the motions of the tag can interfere severely with the observation of protein dynamics. The two-point attached CLaNP-5 lanthanide tag was linked to adenylate kinase. From the paramagnetic relaxation dispersion only motion of the tag is observed. The data can be described accurately by a two-state model in which the protein-attached tag undergoes a 23° tilting motion on a timescale of milliseconds. The work demonstrates the large potential of paramagnetic relaxation dispersion and the challenge to improve current tags to minimize relaxation dispersion from tag movements.


Relaxation dispersion Lanthanide binding tags Protein dynamics Paramagnetic NMR Caged lanthanide NMR probe Adenylate kinase 



Financial support was provided by the Netherlands Organisation for Scientific Research grants 700.10.407 (M.A.S.H) and 700.58.441 (M.U., W.M.L. and J.T.S.), the Howard Hughes Medical Institute and the Office of Basic Energy Sciences, Catalysis Science Program, U.S. Dept. of Energy, award DE-FG02-05ER15699 and National Institutes of Health, award GM100966-01 (R.V.A., L.A.P., R.O. and D.K.), and R.O. is a HHMI Fellow of the Damon Runyon Cancer Research Foundation, DRG-2114-12.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

10858_2014_9894_MOESM1_ESM.pdf (3.9 mb)
Supplementary material 1 (PDF 4003 kb)


  1. Aime S, Botta M, Ermondi G (1992) NMR study of solution structures and dynamics of lanthanide(III) complexes of DOTA. Inorg Chem 31:4291–4299CrossRefGoogle Scholar
  2. Auer R, Neudecker P, Muhandiram D, Lundstrom P, Hansen D, Konrat R, Kay LE (2009) Measuring the signs of 1Hα chemical shift differences between ground and excited protein states by off-resonance spin-lock R NMR spectroscopy. J Am Chem Soc 131:10832–10833CrossRefGoogle Scholar
  3. Auer R, Hansen D, Neudecker P, Korzhnev DM, Muhandiram D, Konrat R, Kay LE (2010) Measurement of signs of chemical shift differences between ground and excited protein states: a comparison between H(S/M)QC and R methods. J Biomol NMR 46:205–216CrossRefGoogle Scholar
  4. Bertini I, Luchinat C, Parigi G (2002) Magnetic susceptibility in paramagnetic NMR. Progr Nucl Magn Reson Spect 40:249–273CrossRefGoogle Scholar
  5. Bertini I, Calderone V, Cerofolini L, Fragai M, Geraldes CFGC, Hermann P, Luchinat C, Parigi G, Teixeira JMC (2012) The catalytic domain of MMP-1 studied through tagged lanthanides. FEBS Lett 586:557–567CrossRefGoogle Scholar
  6. Bouvignies G, Markwick P, Bruscheweiler R, Blackledge M (2006) Simultaneous determination of protein backbone structure and dynamics from residual dipolar couplings. J Am Chem Soc 128:15100–15101CrossRefGoogle Scholar
  7. Bouvignies G, Vallurupalli P, Hansen DF, Correia BE, Lange O, Bah A, Vernon RM, Dahlquist FW, Baker D, Kay LE (2011) Solution structure of a minor and transiently formed state of a T4 lysozyme mutant. Nature 477:111–114CrossRefADSGoogle Scholar
  8. Camacho-Zarco AR, Munari F, Wegstroth M, Liu WM, Ubbink M, Becker S, Zweckstetter M (2015) Multiple paramagnetic effects through a tagged reporter protein. Angew Chem Int Ed 54:336–339Google Scholar
  9. Carver JP, Richards RE (1972) A general two-site solution for the chemical exchange produced dependence of T2 upon the Carr-Purcell pulse separation. J Magn Reson 6:89–105ADSGoogle Scholar
  10. Cornilescu G, Marquardt JL, Ottiger M, Bax A (1998) Validation of protein structure from anisotropic carbonyl chemical shifts in a dilute liquid crystalline phase. J Am Chem Soc 120:6836–6837CrossRefGoogle Scholar
  11. Dasgupta S, Hu XY, Keizers PHJ, Liu WM, Luchinat C, Nagulapalli M, Overhand M, Parigi G, Sgheri L, Ubbink M (2011) Narrowing the conformational space sampled by two-domain proteins with paramagnetic probes in both domains. J Biomol NMR 51:253–263CrossRefGoogle Scholar
  12. Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, Bax A (1995) NMRPipe—A multidimensional spectral processing systeme based on UNIX pipes. J Biomol NMR 6:277–293CrossRefGoogle Scholar
  13. Eichmueller C, Skrynnikov NR (2007) Observation of μs time-scale protein dynamics in the presence of Ln(3+) ions: application to the N-terminal domain of cardiac troponin C. J Biomol NMR 37:79–95CrossRefGoogle Scholar
  14. Grey MJ, Wang CY, Palmer AG (2003) Disulfide bond isomerization in basic pancreatic trypsin inhibitor: multisite chemical exchange quantified by CPMG relaxation dispersion and chemical shift modeling. J Am Chem Soc 125:14324–14335CrossRefGoogle Scholar
  15. Guan JY, Keizers PHJ, Liu WM, Lohr F, Skinner SP, Heeneman EA, Schwalbe H, Ubbink M, Siegal G (2013) Small-molecule binding sites on proteins established by paramagnetic NMR spectroscopy. J Am Chem Soc 135:5859–5868CrossRefGoogle Scholar
  16. Hass MAS, Keizers PHJ, Blok A, Hiruma Y, Ubbink M (2010) Validation of a lanthanide tag for the analysis of protein dynamics by paramagnetic NMR spectroscopy. J Am Chem Soc 132:9952–9953CrossRefGoogle Scholar
  17. Haussinger D, Huang JR, Grzesiek S (2009) DOTA-M8: an extremely rigid, high-affinity lanthanide chelating tag for PCS NMR spectroscopy. J Am Chem Soc 131:14761–14767CrossRefGoogle Scholar
  18. Henzler-Wildman KA, Thai V, Lei M, Ott M, Wolf-Watz M, Fenn T, Pozharski E, Wilson MA, Petsko GA, Karplus M, Hubner CG, Kern D (2007a) Intrinsic motions along an enzymatic reaction trajectory. Nature 450:838–844CrossRefADSGoogle Scholar
  19. Henzler-Wildman KA, Lei M, Thai V, Kerns SJ, Karplus M, Kern D (2007b) A hierarchy of timescales in protein dynamics is linked to enzyme catalysis. Nature 450:913–916CrossRefADSGoogle Scholar
  20. Hiruma Y, Hass MAS, Kikui Y, Liu WM, Olmez B, Skinner SP, Blok A, Kloosterman A, Koteishi H, Lohr F, Schwalbe H, Nojiri M, Ubbink M (2013) The structure of the cytochrome P450cam-putidaredoxin complex determined by paramagnetic NMR spectroscopy and crystallography. J Mol Biol 425:4353–4365CrossRefGoogle Scholar
  21. John M, Park AY, Pintacuda G, Dixon NE, Otting G (2005) Weak alignment of paramagnetic proteins warrants correction for residual CSA effects in measurements of pseudocontact shifts. J Am Chem Soc 127:17190–17191CrossRefGoogle Scholar
  22. Keizers PHJ, Ubbink M (2011) Paramagnetic tagging for protein structure and dynamics analysis. Progr Nucl Magn Reson Spect 58:88–96CrossRefGoogle Scholar
  23. Keizers PHJ, Desreux JF, Overhand M, Ubbink M (2007) Increased paramagnetic effect of a lanthanide protein probe by two-point attachment. J Am Chem Soc 129:9292–9293CrossRefGoogle Scholar
  24. Keizers PHJ, Saragliadis A, Hiruma Y, Overhand M, Ubbink M (2008) Design, synthesis, and evaluation of a lanthanide chelating protein probe: CLaNP-5 yields predictable paramagnetic effects independent of environment. J Am Chem Soc 130:14802–14812CrossRefGoogle Scholar
  25. Keizers PHJ, Mersinli B, Reinle W, Donauer J, Hiruma Y, Hannemann F, Overhand M, Bernhardt R, Ubbink M (2010) A solution model of the complex formed by adrenodoxin and adrenodoxin reductase determined by paramagnetic NMR spectroscopy. Biochemistry 49:6846–6855CrossRefGoogle Scholar
  26. Koehler J, Meiler J (2011) Expanding the utility of NMR restraints with paramagnetic compounds: background and practical aspects. Progr Nucl Magn Reson Spect 59:360–389CrossRefGoogle Scholar
  27. Liu WM, Keizers PHJ, Hass MAS, Blok A, Timmer M, Sarris AJC, Overhand M, Ubbink M (2012) A pH-sensitive, colorful, lanthanide-chelating paramagnetic NMR probe. J Am Chem Soc 134:17306–17313Google Scholar
  28. Markwick PR, Bouvignies G, Salmon L, McCammon J, Nilges M, Blackledge M (2009) Toward a unified representation of protein structural dynamics in solution. J Am Chem Soc 131:16968–16975CrossRefGoogle Scholar
  29. Monod J, Wyman J, Changeux JP (1965) On the nature of allosteric transitions: a plausible model. J Mol Biol 12:88–118CrossRefGoogle Scholar
  30. Natrajan LS, Khoabane NM, Dadds BL, Muryn CA, Pritchard RG, Heath SL, Kenwright AM, Kuprov I, Faulkner S (2010) Probing the structure, conformation, and stereochemical exchange in a family of lanthanide complexes derived from tetrapyridyl-appended cyclen. Inorg Chem 49:7700–7709CrossRefGoogle Scholar
  31. Orekhov VY, Korzhnev DM, Kay LE (2004) Double- and zero-quantum NMR relaxation dispersion experiments sampling millisecond time scale dynamics in proteins. J Am Chem Soc 126:1886–1891CrossRefGoogle Scholar
  32. Otting G (2008) Prospects for lanthanides in structural biology by NMR. J Biomol NMR 42:1–9CrossRefGoogle Scholar
  33. Palmer AG, Kroenke CD, Loria JP (2001) Nuclear magnetic resonance methods for quantifying microsecond-to-millisecond motions in biological macromolecules. Methods Enzymol 339:204–238CrossRefGoogle Scholar
  34. Pervushin K, Riek R, Wider G, Wuthrich K (1997) Attenuated T2 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 94:12366–12371CrossRefADSGoogle Scholar
  35. Schmitz C, John M, Park AY, Dixon NE, Otting G, Pintacuda G, Huber T (2006) Efficient Chi-tensor determination and NH assignment of paramagnetic proteins. J Biomol NMR 35:79–87CrossRefGoogle Scholar
  36. Schmitz C, Stanton-Cook MJ, Su XC, Otting G, Huber T (2008) Numbat: an interactive software tool for fitting Delta Chi-tensors to molecular coordinates using pseudocontact shifts. J Biomol NMR 41:179–189CrossRefGoogle Scholar
  37. Shishmarev D, Otting G (2013) How reliable are pseudocontact shifts induced in proteins and ligands by mobile paramagnetic metal tags? A modelling study. J Biomol NMR 56:203–216CrossRefGoogle Scholar
  38. Skrynnikov NR, Dahlquist FW, Kay LE (2002) Reconstructing NMR spectra of “invisible” excited protein states using HSQC and HMQC experiments. J Am Chem Soc 124:12352–12360CrossRefGoogle Scholar
  39. Tollinger M, Skrynnikov NR, Mulder FAA, Forman-Kay JD, Kay LE (2001) Slow dynamics in folded and unfolded states of an SH3 domain. J Am Chem Soc 123:11341–11352CrossRefGoogle Scholar
  40. Torda AE, Scheek RM, van Gunsteren WF (1990) Time-averaged nuclear Overhauser effect distance restraints applied to tendamistat. J Mol Biol 214:223–235CrossRefGoogle Scholar
  41. Vallurupalli P, Hansen DF, Stollar E, Meirovitch E, Kay LE (2007) Measurement of bond vector orientations in invisible excited states of proteins. Proc Natl Acad Sci USA 104:18473–18477CrossRefADSGoogle Scholar
  42. Vallurupalli P, Hansen DF, Kay LE (2008a) Probing structure in invisible protein states with anisotropic NMR chemical shifts. J Am Chem Soc 130:2734–2735CrossRefGoogle Scholar
  43. Vallurupalli P, Hansen DF, Kay LE (2008b) Structures of invisible, excited protein states by relaxation dispersion NMR spectroscopy. Proc Natl Acad Sci USA 105:11766–11771CrossRefADSGoogle Scholar
  44. Vlasie MD, Comuzzi C, Van den Nieuwendijk AMCH, Prudencio M, Overhand M, Ubbink M (2007) Long-range-distance NMR effects in a protein labeled with a lanthanide-DOTA chelate. Chem Eur J 13:1715–1723CrossRefGoogle Scholar
  45. Vranken WF, Boucher W, Stevens TJ, Fogh RH, Pajon A, Llinas P, Ulrich EL, Markley JL, Ionides J, Laue ED (2005) The CCPN data model for NMR spectroscopy: development of a software pipeline. Prot Struct Funct Bioinform 59:687–696CrossRefGoogle Scholar
  46. Wang X, Srisailam S, Yee AA, Lemak A, Arrowsmith C, Prestegard JH, Tian F (2007) Domain-domain motions in proteins from time-modulated pseudocontact shifts. J Biomol NMR 39:53–61CrossRefGoogle Scholar
  47. Wolf-Watz M, Thai V, Henzler-Wildman K, Hadjipavlou G, Eisenmesser EZ, Kern D (2004) Linkage between dynamics and catalysis in a thermophilic-mesophilic enzyme pair. Nat Struct Mol Biol 11:945–949CrossRefGoogle Scholar
  48. Xu XF, Keizers PHJ, Reinle W, Hannemann F, Bernhardt R, Ubbink M (2009) Intermolecular dynamics studied by paramagnetic tagging. J Biomol NMR 43:247–254CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2015

Authors and Affiliations

  • Mathias A. S. Hass
    • 1
  • Wei-Min Liu
    • 1
  • Roman V. Agafonov
    • 2
  • Renee Otten
    • 2
  • Lien A. Phung
    • 2
  • Jesika T. Schilder
    • 1
  • Dorothee Kern
    • 2
  • Marcellus Ubbink
    • 1
    Email author
  1. 1.Leiden Institute of ChemistryLeiden UniversityLeidenThe Netherlands
  2. 2.Department of Biochemistry, Howard Hughes Medical InstituteBrandeis UniversityWalthamUSA

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