Journal of Biomolecular NMR

, Volume 63, Issue 3, pp 275–282 | Cite as

Encoded loop-lanthanide-binding tags for long-range distance measurements in proteins by NMR and EPR spectroscopy

  • Dominic Barthelmes
  • Markus Gränz
  • Katja Barthelmes
  • Karen N. Allen
  • Barbara Imperiali
  • Thomas PrisnerEmail author
  • Harald SchwalbeEmail author


We recently engineered encodable lanthanide binding tags (LBTs) into proteins and demonstrated their applicability in Nuclear Magnetic Resonance (NMR) spectroscopy, X-ray crystallography and luminescence studies. Here, we engineered two-loop-LBTs into the model protein interleukin-1β (IL1β) and measured 1H, 15N-pseudocontact shifts (PCSs) by NMR spectroscopy. We determined the Δχ-tensors associated with each Tm3+-loaded loop-LBT and show that the experimental PCSs yield structural information at the interface between the two metal ion centers at atomic resolution. Such information is very valuable for the determination of the sites of interfaces in protein–protein-complexes. Combining the experimental PCSs of the two-loop-LBT construct IL1β-S2R2 and the respective single-loop-LBT constructs IL1β-S2, IL1β-R2 we additionally determined the distance between the metal ion centers. Further, we explore the use of two-loop LBTs loaded with Gd3+ as a novel tool for distance determination by Electron Paramagnetic Resonance spectroscopy and show the NMR-derived distances to be remarkably consistent with distances derived from Pulsed Electron–Electron Dipolar Resonance.


Paramagnetic NMR EPR PELDOR Lanthanide binding tags 



We thank Dmitry Akhmetzyanov for helpful discussions. This work was supported by Deutsche Forschungsgemeinschaft (DFG) in collaborative research centers 807 and 902. H.S. and T.P. are members of the DFG-funded cluster of excellence: macromolecular complexes and BMRZ is supported by the state of Hesse. K.N.A and B.I. acknowledge the support of NSF Grant MCB 0744415.

Supplementary material

10858_2015_9984_MOESM1_ESM.docx (1012 kb)
Supplementary material 1 (DOCX 1013 kb)


  1. Alonso-García N, García-Rubio I, Manso JA et al (2015) Combination of X-ray crystallography, SAXS and DEER to obtain the structure of the FnIII-3,4 domains of integrin α6β4. Acta Crystallogr D Biol Crystallogr 71:969–985. doi: 10.1107/S1399004715002485 CrossRefGoogle Scholar
  2. Barthelmes K, Reynolds AM, Peisach E et al (2011) Engineering encodable lanthanide-binding tags into loop regions of proteins. J Am Chem Soc 133:808–819. doi: 10.1021/ja104983t CrossRefGoogle Scholar
  3. Bentrop D, Bertini I, Cremonini MA et al (1997) Solution structure of the paramagnetic complex of the N-terminal domain of calmodulin with two Ce3 + ions by 1H NMR. Biochemistry 36:11605–11618. doi: 10.1021/bi971022+ CrossRefGoogle Scholar
  4. Bernstein FC, Koetzle TF, Williams GJ et al (1977) The Protein Data Bank. A computer-based archival file for macromolecular structures. Eur J Biochem 80:319–324. doi: 10.1016/S0022-2836(77)80200-3 CrossRefGoogle Scholar
  5. Bertini I, Janik MB, Lee YM et al (2001) Magnetic susceptibility tensor anisotropies for a lanthanide ion series in a fixed protein matrix. J Am Chem Soc 123:4181–4188CrossRefGoogle Scholar
  6. Bertini I, Luchinat C, Parigi G (2002) Magnetic susceptibility in paramagnetic NMR. Prog Nucl Magn Reson Spectrosc 40:249–273. doi: 10.1016/S0079-6565(02)00002-X CrossRefGoogle Scholar
  7. Dalaloyan A, Qi M, Ruthstein S et al (2015) Gd(iii)–Gd(iii) EPR distance measurements—the range of accessible distances and the impact of zero field splitting. Phys Chem Chem Phys 17:18464–18476. doi: 10.1039/C5CP02602D CrossRefGoogle Scholar
  8. Duss O, Michel E, Yulikov M et al (2014) Structural basis of the non-coding RNA RsmZ acting as a protein sponge. Nature 509:588–592. doi: 10.1038/nature13271 CrossRefADSGoogle Scholar
  9. Duss O, Yulikov M, Allain FH, Jeschke G (2015) Combining NMR and EPR to determine structures of large RNAs and protein–RNA complexes in solution. Methods Enzymol 558:279–331. doi: 10.1016/bs.mie.2015.02.005 CrossRefGoogle Scholar
  10. Garbuio L, Bordignon E, Brooks EK et al (2013) Orthogonal Spin Labeling and Gd(III)–nitroxide distance measurements on bacteriophage T4-lysozyme. J Phys Chem B 117:3145–3153. doi: 10.1021/jp401806g CrossRefGoogle Scholar
  11. Göbl C, Madl T, Simon B, Sattler M (2014) NMR approaches for structural analysis of multidomain proteins and complexes in solution. Prog Nucl Magn Reson Spectrosc 80:26–63. doi: 10.1016/j.pnmrs.2014.05.003 CrossRefGoogle Scholar
  12. Goldfarb D (2014) Gd3+ spin labeling for distance measurements by pulse EPR spectroscopy. Phys Chem Chem Phys 16:9685. doi: 10.1039/c3cp53822b CrossRefGoogle Scholar
  13. Gordon-Grossman M, Kaminker I, Gofman Y et al (2011) W-Band pulse EPR distance measurements in peptides using Gd3+–dipicolinic acid derivatives as spin labels. Phys Chem Chem Phys 13:10771. doi: 10.1039/c1cp00011j CrossRefGoogle Scholar
  14. Hass MA, Ubbink M (2014) Structure determination of protein–protein complexes with long-range anisotropic paramagnetic NMR restraints. Curr Opin Struct Biol 24:45–53. doi: 10.1016/ CrossRefGoogle Scholar
  15. Jeschke G, Chechik V, Ionita P et al (2006) DeerAnalysis2006—a comprehensive software package for analyzing pulsed ELDOR data. Appl Magn Reson 30:473–498. doi: 10.1007/BF03166213 CrossRefGoogle Scholar
  16. Keizers PHJ, Ubbink M (2011) Paramagnetic tagging for protein structure and dynamics analysis. Prog Nucl Magn Reson Spectrosc 58:88–96. doi: 10.1016/j.pnmrs.2010.08.001 CrossRefGoogle Scholar
  17. Lapinaite A, Simon B, Skjaerven L et al (2013) The structure of the box C/D enzyme reveals regulation of RNA methylation. Nature 502:519–523. doi: 10.1038/nature12581 CrossRefADSGoogle Scholar
  18. Loscha KV, Herlt AJ, Qi R et al (2012) Multiple-site labeling of proteins with unnatural amino acids. Angew Chem Int Ed 51:2243–2246. doi: 10.1002/anie.201108275 CrossRefGoogle Scholar
  19. Lueders P, Jeschke G, Yulikov M (2011) Double electron–electron resonance measured between Gd3+ ions and nitroxide radicals. J Phys Chem Lett 2:604–609. doi: 10.1021/jz200073h CrossRefGoogle Scholar
  20. Lueders P, Jäger H, Hemminga MA et al (2013) Distance measurements on orthogonally spin-labeled membrane spanning WALP23 polypeptides. J Phys Chem B 117:2061–2068. doi: 10.1021/jp311287t CrossRefGoogle Scholar
  21. Lynch M (2013) Evolutionary diversification of the multimeric states of proteins. Proc Natl Acad Sci U S A 110:E2821–E2828. doi: 10.1073/pnas.1310980110 CrossRefADSGoogle Scholar
  22. Mackereth CD, Madl T, Bonnal S et al (2011) Multi-domain conformational selection underlies pre-mRNA splicing regulation by U2AF. Nature 475:408–411. doi: 10.1038/nature10171 CrossRefGoogle Scholar
  23. Martin LJ, Hähnke MJ, Nitz M et al (2007) Double-lanthanide-binding tags: design, photophysical properties, and NMR applications. J Am Chem Soc 129:7106–7113. doi: 10.1021/ja070480v CrossRefGoogle Scholar
  24. Matalon E, Huber T, Hagelueken G et al (2013) Gadolinium(III) spin labels for high-sensitivity distance measurements in transmembrane helices. Angew Chem Int Ed 52:11831–11834. doi: 10.1002/anie.201305574 CrossRefGoogle Scholar
  25. Pannier M, Veit S, Godt A et al (2000) Dead-time free measurement of dipole–dipole interactions between electron spins. J Magn Reson 142:331–340. doi: 10.1006/jmre.1999.1944 CrossRefADSGoogle Scholar
  26. Potapov A, Song Y, Meade TJ et al (2010) Distance measurements in model bis-Gd(III) complexes with flexible “bridge”. Emulation of biological molecules having flexible structure with Gd(III) labels attached. J Magn Reson 205:38–49. doi: 10.1016/j.jmr.2010.03.019 CrossRefADSGoogle Scholar
  27. Raitsimring AM, Gunanathan C, Potapov A et al (2007) Gd3+ complexes as potential spin labels for high field pulsed EPR distance measurements. J Am Chem Soc 129:14138–14139. doi: 10.1021/ja075544g CrossRefGoogle Scholar
  28. Russo L, Maestre-Martinez M, Wolff S et al (2013) Interdomain dynamics explored by paramagnetic NMR. J Am Chem Soc 135:17111–17120. doi: 10.1021/ja408143f CrossRefGoogle Scholar
  29. Schiemann O, Prisner TF (2007) Long-range distance determinations in biomacromolecules by EPR spectroscopy. Q Rev Biophys 40:1. doi: 10.1017/S003358350700460X CrossRefGoogle Scholar
  30. Schmitz C, Stanton-Cook MJ, Su X-C et al (2008) Numbat: an interactive software tool for fitting Deltachi-tensors to molecular coordinates using pseudocontact shifts. J Biomol NMR 41:179–189. doi: 10.1007/s10858-008-9249-z CrossRefGoogle Scholar
  31. Silvaggi NR, Martin LJ, Schwalbe H et al (2007) Double-lanthanide-binding tags for macromolecular crystallographic structure determination. J Am Chem Soc 129:7114–7120. doi: 10.1021/ja070481n CrossRefGoogle Scholar
  32. Song Y, Meade TJ, Astashkin AV et al (2011) Pulsed dipolar spectroscopy distance measurements in biomacromolecules labeled with Gd(III) markers. J Magn Reson 210:59–68. doi: 10.1016/j.jmr.2011.02.010 CrossRefADSGoogle Scholar
  33. Stoll S, Schweiger A (2006) EasySpin, a comprehensive software package for spectral simulation and analysis in EPR. J Magn Reson 178:42–55. doi: 10.1016/j.jmr.2005.08.013 CrossRefADSGoogle Scholar
  34. Studier FW (2005) Protein production by auto-induction in high density shaking cultures. Protein Expr Purif 41:207–234. doi: 10.1016/j.pep.2005.01.016 CrossRefGoogle Scholar
  35. Tamm LK, Lai AL, Li Y (2007) Combined NMR and EPR spectroscopy to determine structures of viral fusion domains in membranes. Biochim Biophys Acta Biomembr 1768:3052–3060. doi: 10.1016/j.bbamem.2007.09.010 CrossRefGoogle Scholar
  36. Wöhnert J, Franz KJ, Nitz M et al (2003) Protein alignment by a coexpressed lanthanide-binding tag for the measurement of residual dipolar couplings. J Am Chem Soc 125:13338–13339. doi: 10.1021/ja036022d CrossRefGoogle Scholar
  37. Wolfram Research Inc., (2014) Mathematica version 10.0. ChampaignGoogle Scholar
  38. Yagi H, Banerjee D, Graham B et al (2011) Gadolinium tagging for high-precision measurements of 6 nm distances in protein assemblies by EPR. J Am Chem Soc 133:10418–10421. doi: 10.1021/ja204415w CrossRefGoogle Scholar
  39. Yulikov M, Lueders P, Farooq Warsi M et al (2012) Distance measurements in Au nanoparticles functionalized with nitroxide radicals and Gd3+–DTPA chelate complexes. Phys Chem Chem Phys 14:10732. doi: 10.1039/c2cp40282c CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2015

Authors and Affiliations

  • Dominic Barthelmes
    • 1
  • Markus Gränz
    • 2
  • Katja Barthelmes
    • 1
    • 5
  • Karen N. Allen
    • 3
  • Barbara Imperiali
    • 4
  • Thomas Prisner
    • 2
    Email author
  • Harald Schwalbe
    • 1
    Email author
  1. 1.Institute of Organic Chemistry and Chemical Biology, Center for Biomolecular Magnetic ResonanceGoethe University FrankfurtFrankfurt Am MainGermany
  2. 2.Institute of Physical and Theoretical Chemistry, Center for Biomolecular Magnetic ResonanceGoethe University FrankfurtFrankfurt Am MainGermany
  3. 3.Department of ChemistryBoston UniversityBostonUSA
  4. 4.Departments of Chemistry and BiologyMassachusetts Institute of TechnologyCambridgeUSA
  5. 5.Department of Chemistry, Munich Center for Integrated Protein Science and Chair Biomolecular NMRTechnical University MunichGarchingGermany

Personalised recommendations