Journal of Biomolecular NMR

, Volume 58, Issue 3, pp 149–154 | Cite as

A decadentate Gd(III)-coordinating paramagnetic cosolvent for protein relaxation enhancement measurement

  • Xin-Hua Gu
  • Zhou Gong
  • Da-Chuan Guo
  • Wei-Ping ZhangEmail author
  • Chun TangEmail author


Solvent paramagnetic relaxation enhancement (sPRE) arises from random collisions between paramagnetic cosolvent and protein of interest. The sPRE can be readily measured, affording protein structure information. However, lack of an inert cosolvent probe may yield sPRE values that are not consistent with protein structure. Here we synthesized a new sPRE probe, triethylenetetraamine hexaacetate trimethylamide gadolinium, or Gd(III)–TTHA–TMA. With a total of 10 coordination sites, this paramagnetic cosovlent eliminates an inner-sphere water molecule that can otherwise transfer relaxation to protein through exchange. With the metal ion centered, the new probe is largely spherical with a radius of 4.0 Å, permitting accurate back calculation of sPRE. The effectiveness Gd(III)–TTHA–TMA as a sPRE probe was demonstrated on three well-studied protein systems.


NMR spectroscopy Protein structures Paramagnetic relaxation enhancement Paramagnetic probe Solvent PRE 



The work was supported by funds from the Ministry of Science and Technology of China (2013CB910200), the National Natural Sciences Foundation of China (21073230, 31170728 and 31125007), and Zhejiang Provincial Natural Science Foundation of China (Z2110059). C.T. is an international early career scientist of Howard Hughes Medical Institute (HHMI).

Supplementary material

10858_2014_9817_MOESM1_ESM.docx (896 kb)
Supplementary material 1 (DOCX 897 kb)


  1. Berardi MJ, Shih WM, Harrison SC, Chou JJ (2011) Mitochondrial uncoupling protein 2 structure determined by NMR molecular fragment searching. Nature 476:109–113CrossRefGoogle Scholar
  2. Bernini A, Venditti V, Spiga O, Niccolai N (2009) Probing protein surface accessibility with solvent and paramagnetic molecules. Prog Nucl Magn Reson Spectrosc 54:278–289CrossRefGoogle Scholar
  3. Caravan P, Ellison JJ, McMurry TJ, Lauffer RB (1999) Gadolinium(III) chelates as MRI contrast agents: structure, dynamics, and applications. Chem Rev 99:2293–2352CrossRefGoogle Scholar
  4. Clore GM, Iwahara J (2009) Theory, practice, and applications of paramagnetic relaxation enhancement for the characterization of transient low-population states of biological macromolecules and their complexes. Chem Rev 109:4108–4139CrossRefGoogle Scholar
  5. Derrick JP, Wigley DB (1994) The third IgG-binding domain from streptococcal protein G. An analysis by X-ray crystallography of the structure alone and in a complex with Fab. J Mol Biol 243:906–918CrossRefGoogle Scholar
  6. Eliezer D (2012) Distance information for disordered proteins from NMR and ESR measurements using paramagnetic spin labels. Methods Mol Biol 895:127–138CrossRefGoogle Scholar
  7. Feese MD, Comolli L, Meadow ND, Roseman S, Remington SJ (1997) Structural studies of the Escherichia coli signal transducing protein IIAGlc: implications for target recognition. Biochemistry 36:16087–16096CrossRefGoogle Scholar
  8. Gillespie JR, Shortle D (1997) Characterization of long-range structure in the denatured state of staphylococcal nuclease. I. Paramagnetic relaxation enhancement by nitroxide spin labels. J Mol Biol 268:158–169CrossRefGoogle Scholar
  9. Gottstein D, Reckel S, Dotsch V, Guntert P (2012) Requirements on paramagnetic relaxation enhancement data for membrane protein structure determination by NMR. Structure 20:1019–1027CrossRefGoogle Scholar
  10. Gronenborn AM, Filpula DR, Essig NZ, Achari A, Whitlow M, Wingfield PT, Clore GM (1991) A novel, highly stable fold of the immunoglobulin binding domain of streptococcal protein G. Science 253:657–661ADSCrossRefGoogle Scholar
  11. Guttler T, Madl T, Neumann P, Deichsel D, Corsini L, Monecke T, Ficner R, Sattler M, Gorlich D (2010) NES consensus redefined by structures of PKI-type and Rev-type nuclear export signals bound to CRM1. Nat Struct Mol Biol 17:1367–1376CrossRefGoogle Scholar
  12. Hernandez G, Teng CL, Bryant RG, LeMaster DM (2002) O2 penetration and proton burial depth in proteins: applicability to fold family recognition. J Am Chem Soc 124:4463–4472CrossRefGoogle Scholar
  13. Iwahara J, Schwieters CD, Clore GM (2004) Ensemble approach for NMR structure refinement against (1)H paramagnetic relaxation enhancement data arising from a flexible paramagnetic group attached to a macromolecule. J Am Chem Soc 126:5879–5896CrossRefGoogle Scholar
  14. Iwahara J, Zweckstetter M, Clore GM (2006) NMR structural and kinetic characterization of a homeodomain diffusing and hopping on nonspecific DNA. Proc Natl Acad Sci USA 103:15062–15067ADSCrossRefGoogle Scholar
  15. Iwahara J, Tang C, Clore GM (2007) Practical aspects of 1H transverse paramagnetic relaxation enhancement measurements on macromolecules. J Magn Reson 184:185–195ADSCrossRefGoogle Scholar
  16. Karaca E, Bonvin AM (2013) Advances in integrative modeling of biomolecular complexes. Methods 59:372–381CrossRefGoogle Scholar
  17. Keizers PH, 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
  18. Klammt C, Maslennikov I, Bayrhuber M, Eichmann C, Vajpai N, Chiu EJ, Blain KY, Esquivies L, Kwon JH, Balana B, Pieper U, Sali A, Slesinger PA, Kwiatkowski W, Riek R, Choe S (2012) Facile backbone structure determination of human membrane proteins by NMR spectroscopy. Nat Methods 9:834–839CrossRefGoogle Scholar
  19. Koehler J, Meiler J (2011) Expanding the utility of NMR restraints with paramagnetic compounds: background and practical aspects. Prog Nucl Magn Reson Spectrosc 59:360–389CrossRefGoogle Scholar
  20. Madl T, Bermel W, Zangger K (2009) Use of relaxation enhancements in a paramagnetic environment for the structure determination of proteins using NMR spectroscopy. Angew Chem Int Ed Engl 48:8259–8262CrossRefGoogle Scholar
  21. Madl T, Guttler T, Gorlich D, Sattler M (2011) Structural analysis of large protein complexes using solvent paramagnetic relaxation enhancements. Angew Chem Int Ed Engl 50:3993–3997CrossRefGoogle Scholar
  22. Mal TK, Ikura M, Kay LE (2002) The ATCUN domain as a probe of intermolecular interactions: application to calmodulin-peptide complexes. J Am Chem Soc 124:14002–14003CrossRefGoogle Scholar
  23. Otting G (2010) Protein NMR using paramagnetic ions. Annu Rev Biophys 39:387–405CrossRefGoogle Scholar
  24. Petros AM, Mueller L, Kopple KD (1990) NMR identification of protein surfaces using paramagnetic probes. Biochemistry 29:10041–10048CrossRefGoogle Scholar
  25. 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
  26. Quiocho FA, Spurlino JC, Rodseth LE (1997) Extensive features of tight oligosaccharide binding revealed in high-resolution structures of the maltodextrin transport/chemosensory receptor. Structure 5:997–1015CrossRefGoogle Scholar
  27. Schwieters CD, Kuszewski JJ, Clore GM (2006) Using Xplor-NIH for NMR molecular structure determination. Prog Nucl Magn Reson Spectrosc 48:47–62CrossRefGoogle Scholar
  28. Solomon I (1955) Relaxation processes in a system of two spins. Phys Rev 99:559–565ADSCrossRefGoogle Scholar
  29. Solomon I, Bloembergen N (1956) Nuclear magnetic interactions in the Hf molecule. J Chem Phys 25:261–266ADSCrossRefGoogle Scholar
  30. Venditti V, Niccolai N, Butcher SE (2008) Measuring the dynamic surface accessibility of RNA with the small paramagnetic molecule TEMPOL. Nucleic Acids Res 36:e20CrossRefGoogle Scholar
  31. Wang G, Louis JM, Sondej M, Seok YJ, Peterkofsky A, Clore GM (2000) Solution structure of the phosphoryl transfer complex between the signal transducing proteins HPr and IIA(glucose) of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system. EMBO J 19:5635–5649CrossRefGoogle Scholar
  32. Wang Y, Schwieters CD, Tjandra N (2012) Parameterization of solvent-protein interaction and its use on NMR protein structure determination. J Magn Reson 221:76–84ADSCrossRefGoogle Scholar
  33. Wüthrich KW (1986) NMR of proteins and nucleic acids. Wiley, New YorkGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  1. 1.State Key Laboratory of Magnetic Resonance and Atomic Molecular Physics and Wuhan Institute of Physics and MathematicsChinese Academy of SciencesWuhanChina
  2. 2.Department of PharmacologyZhejiang University School of MedicineHangzhouChina

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