Paramagnetic-iterative relaxation matrix approach: extracting PRE-restraints from NOESY spectra for 3D structure elucidation of biomolecules

  • E. C. Cetiner
  • H. R. A. Jonker
  • C. Helmling
  • D. B. Gophane
  • C. Grünewald
  • S. Th. Sigurdsson
  • H. SchwalbeEmail author


Paramagnetic relaxation enhancement (PRE) can be used to determine long-range distance restraints in biomolecules. The PREs are typically determined by analysis of intensity differences in HSQC experiments of paramagnetic and diamagnetic spin labels. However, this approach requires both isotope- and spin-labelling. Herein, we report a novel method to evaluate NOESY intensities in the presence of a paramagnetic moiety to determine PRE restraints. The advantage of our approach over HSQC-based approaches is the increased number of available signals without the need for isotope labelling. NOESY intensities affected by a paramagnetic center were evaluated during a structure calculation within the paramagnetic iterative relaxation matrix approach (P-IRMA). We applied P-IRMA to a 14-mer RNA with a known NMR solution structure, which allowed us to assess the quality of the PRE restraints. To this end, three different spin labels have been attached at different positions of the 14-mer to test the influence of flexibility on the structure calculation. Structural disturbances introduced by the spin label have been evaluated by chemical shift analysis. Furthermore, the impact of P-IRMA on the quality of the structure bundles were tested by intentionally leaving out available diamagnetic restraints. Our analyses show that P-IRMA is a powerful tool to refine RNA structures for systems that are insufficiently described by using only diamagnetic restraints.


RNA IRMA PRE NMR Paramagnetic Spin label NOESY 



E.C.C. gratefully acknowledges a MainCampus scholarship provided by the Stiftung Polytechnische Gesellschaft (Frankfurt). We would like to thank E. Stirnal, M. Gränz and E. Jaumann for help with experiments. The work was supported by DFG through CRC902 and by the state of Hesse through institutional funding to BMRZ.

Supplementary material

10858_2019_282_MOESM1_ESM.docx (2.4 mb)
Supplementary material 1 (DOCX 2462 kb)


  1. Azarkh M, Singh V, Okle O, Seemann IT, Dietrich DR, Hartig JS, Drescher M (2013) Site-directed spin-labeling of nucleotides and the use of in-cell EPR to determine long-range distances in a biologically relevant environment. Nat Protoc 8:131–147CrossRefGoogle Scholar
  2. Battiste JL, Wagner G (2000) Utilization of site-directed spin labeling and high-resolution heteronuclear nuclear magnetic resonance for global fold determination of large proteins with limited nuclear overhauser effect data†. Biochemistry 39:5355–5365CrossRefGoogle Scholar
  3. Beswick V, Guerois R, Cordier-Ochsenbein F, Coïc Y-M, Huynh-Dinh T, Tostain J, Noël J-P, Sanson A, Neumann J-M (1998) Dodecylphosphocholine micelles as a membrane-like environment: new results from NMR relaxation and paramagnetic relaxation enhancement analysis. Eur Biophys J 28:48–58CrossRefGoogle Scholar
  4. Boelens R, Koning TMG, Kaptein R (1988) Determination of biomolecular structures from proton-proton NOE’s using a relaxation matrix approach. J Mol Struct 173:299–311ADSCrossRefGoogle Scholar
  5. Boelens R, Koning TMG, van der Marel GA, van Boom JH, Kaptein R (1989) Iterative procedure for structure determination from proton-proton NOEs using a full relaxation matrix approach. Application to a DNA octamer. J Magn Reson 82:290–308ADSGoogle Scholar
  6. Brueschweiler R, Roux B, Blackledge M, Griesinger C, Karplus M, Ernst RR (1992) Influence of rapid intramolecular motion on NMR cross-relaxation rates. A molecular dynamics study of antamanide in solution. J Am Chem Soc 114:2289–2302CrossRefGoogle Scholar
  7. Brünger AT, Adams PD, Clore GM, DeLano WL, Gros P, Grosse-Kunstleve RW, Jiang JS, Kuszewski J, Nilges M, Pannu NS et al (1998) Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr D 54:905–921CrossRefGoogle Scholar
  8. Büttner L, Seikowski J, Wawrzyniak K, Ochmann A, Höbartner C (2013) Synthesis of spin-labeled riboswitch RNAs using convertible nucleosides and DNA-catalyzed RNA ligation. Bioorg Med Chem 21:6171–6180CrossRefGoogle Scholar
  9. Dedmon MM, Lindorff-Larsen K, Christodoulou J, Vendruscolo M, Dobson CM (2005) Mapping long-range interactions in α-synuclein using spin-label NMR and ensemble molecular dynamics simulations. J Am Chem Soc 127:476–477CrossRefGoogle Scholar
  10. Duchardt E, Schwalbe H (2005) Residue specific ribose and nucleobase dynamics of the cUUCGg RNA tetraloop motif by MNMR 13C relaxation. J Biomol NMR 32:295–308CrossRefGoogle Scholar
  11. Edwards TE, Sigurdsson ST (2007) Site-specific incorporation of nitroxide spin-labels into 2′-positions of nucleic acids. Nat Protoc 2:1954–1962CrossRefGoogle Scholar
  12. Fürtig B, Richter C, Bermel W, Schwalbe H (2004) New NMR experiments for RNA nucleobase resonance assignment and chemical shift analysis of an RNA UUCG tetraloop. J Biomol NMR 28:69–79CrossRefGoogle Scholar
  13. Grünewald C, Kwon T, Piton N, Förster U, Wachtveitl J, Engels JW (2008) RNA as scaffold for pyrene excited complexes. Bioorg Med Chem 16:19–26CrossRefGoogle Scholar
  14. Helmling C, Bessi I, Wacker A, Schnorr KA, Jonker HRA, Richter C, Wagner D, Kreibich M, Schwalbe H (2014) Noncovalent spin labeling of riboswitch rnas to obtain long-range structural NMR restraints. ACS Chem Biol 9:1330–1339CrossRefGoogle Scholar
  15. Höbartner C, Sicoli G, Wachowius F, Gophane DB, Sigurdsson STh (2012) Synthesis and characterization of RNA containing a rigid and nonperturbing cytidine-derived spin label. J Org Chem 77:7749–7754CrossRefGoogle Scholar
  16. Iwahara J, Tang C, Marius Clore G (2007) Practical aspects of 1H transverse paramagnetic relaxation enhancement measurements on macromolecules. J Magn Reson 184:185–195ADSCrossRefGoogle Scholar
  17. Kalk A, Berendsen HJC (1976) Proton magnetic relaxation and spin diffusion in proteins. J Magn Reson 1969(24):343–366ADSGoogle Scholar
  18. Kellner R, Mangels C, Schweimer K, Prasch SJ, Weiglmeier PR, Rösch P, Schwarzinger S (2009) SEMPRE: spectral editing mediated by paramagnetic relaxation enhancement. J Am Chem Soc 131:18016–18017CrossRefGoogle Scholar
  19. Linge JP, O’Donoghue SI, Nilges M (2001) Automated assignment of ambiguous nuclear overhauser effects with ARIA. Methods in Enzymology 339:71–90CrossRefGoogle Scholar
  20. Lipari G, Szabo A (1982) Model-free approach to the interpretation of nuclear magnetic resonance relaxation in macromolecules. 1. Theory and range of validity. J Am Chem Soc 104:4546–4559CrossRefGoogle Scholar
  21. Mackereth CD, Madl T, Bonnal S, Simon B, Zanier K, Gasch A, Rybin V, Valcárcel J, Sattler M (2011) Multi-domain conformational selection underlies pre-mRNA splicing regulation by U2AF. Nature 475:408–411CrossRefGoogle Scholar
  22. Macosko JC, Pio MS, Tinoco I, Shin Y-K (1999) A novel 5′ displacement spin-labeling technique for electron paramagnetic resonance spectroscopy of RNA. RNA 5:1158–1166CrossRefGoogle Scholar
  23. Martin NH, Floyd RM, Woodcock HL, Huffman S, Lee C-K (2008) Computation of through-space NMR shielding effects in aromatic ring π-stacked complexes. J Mol Graph Model 26:1125–1130CrossRefGoogle Scholar
  24. Nozinovic S, Fürtig B, Jonker HRA, Richter C, Schwalbe H (2010) High-resolution NMR structure of an RNA model system: the 14-mer cUUCGg tetraloop hairpin RNA. Nucleic Acids Res 38:683–694CrossRefGoogle Scholar
  25. Ramos A, Varani G (1998) A new method to detect long-range protein–RNA contacts: nMR detection of electron–proton relaxation induced by nitroxide spin-labeled RNA. J Am Chem Soc 120:10992–10993CrossRefGoogle Scholar
  26. Schiemann O, Piton N, Plackmeyer J, Bode BE, Prisner TF, Engels JW (2007) Spin labeling of oligonucleotides with the nitroxide TPA and use of PELDOR, a pulse EPR method, to measure intramolecular distances. Nat Protoc 2:904–923CrossRefGoogle Scholar
  27. Schnorr K, Gophane DB, Helmling C, Cetiner E, Pasemann K, Fürtig B, Wacker A, Qureshi NS, Gränz M, Barthelmes D et al (2017) Impact of spin label rigidity on extent and accuracy of distance information from PRE data. J Biomol NMR 68:53–63CrossRefGoogle Scholar
  28. Seven I, Weinrich T, Gränz M, Grünewald C, Brüß S, Krstić I, Prisner TF, Heckel A, Göbel MW (2014) Photolabile protecting groups for nitroxide spin labels. Eur J Org Chem 2014:4037–4043CrossRefGoogle Scholar
  29. Solomon I (1955) Relaxation processes in a system of two spins. Phys Rev 99:559–565ADSCrossRefGoogle Scholar
  30. Stoll S, Schweiger A (2006) EasySpin, a comprehensive software package for spectral simulation and analysis in EPR. J Magn Reson 178:42–55ADSCrossRefGoogle Scholar
  31. Unger SW, Lecomte JTJ, La Mar GN (1985) The utility of the nuclear overhauser effect for peak assignment and structure elucidation in paramagnetic proteins. J Magn Reson 64:521–526ADSGoogle Scholar
  32. van der Walt S, Colbert SC, Varoquaux G (2011) The numPy array: a structure for efficient numerical computation. Comput Sci Eng 13:22–30CrossRefGoogle Scholar
  33. Volkov AN, Worrall JAR, Holtzmann E, Ubbink M (2006) Solution structure and dynamics of the complex between cytochrome c and cytochrome c peroxidase determined by paramagnetic NMR. Proc Natl Acad Sci 103:18945–18950ADSCrossRefGoogle Scholar
  34. Wunderlich CH, Huber RG, Spitzer R, Liedl KR, Kloiber K, Kreutz C (2013) A novel paramagnetic relaxation enhancement tag for nucleic acids: a tool to study structure and dynamics of RNA. ACS Chem Biol 8:2697–2706CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Institut für Organische Chemie und Chemische Biologie, Zentrum für Biomolekulare Magnetische ResonanzGoethe Universität Frankfurt am MainFrankfurt am MainGermany
  2. 2.Department of Chemistry Science InstituteUniversity of IcelandReykjavikIceland

Personalised recommendations