Applications of NMR-Based PRE and EPR-Based DEER Spectroscopy to Homodimer Chain Exchange Characterization and Structure Determination

  • Yunhuang Yang
  • Theresa A. Ramelot
  • Shuisong Ni
  • Robert M. McCarrick
  • Michael A. Kennedy
Part of the Methods in Molecular Biology book series (MIMB, volume 1091)


The success of homodimer structure determination by conventional solution NMR spectroscopy relies greatly on interchain distance restraints (less than 6 Å) derived from nuclear Overhauser effects (NOEs) obtained from 13C-edited, 12C-filtered NOESY experiments. However, these experiments may fail when the mixed 13C-/12C-homodimer is never significantly populated due to slow homodimer chain exchange. Thus, knowledge of the homodimer chain exchange kinetics can be put to practical use in preparing samples using the traditional NMR method. Here, we described detailed procedures for using paramagnetic resonance enhancements (PREs) and EPR spectroscopy to measure homodimer chain exchange kinetics. In addition, PRE and EPR methods can be combined to provide mid-range (<30 Å) and long-range (17–80 Å) interchain distance restraints for homodimer structure determination as a supplement to short-range intrachain and interchain distance restraints (less than 6 Å) typically obtained from 1H-1H NOESY experiments. We present a summary of how to measure these distances using NMR-based PREs and EPR-based double electron electron resonance (DEER) measurements and how to include them in homodimer structure calculations.

Key words

NMR EPR Spectroscopy Homodimer Chain exchange Structure determination 



This work was supported by the National Institute of General Medical Sciences, Grant Number: U54-GM074958; National Science Foundation, Grant Number CHE-0645709; BrukerBiospin, Miami University and Ohio Board of Reagents.


  1. 1.
    Wüthrich K (1986) NMR of proteins and nucleic acids. Wiley, New YorkGoogle Scholar
  2. 2.
    Goodsell DS, Olson AJ (2000) Structural symmetry and protein function. Annu Rev Biophys Biomol Struct 29:105–153CrossRefPubMedGoogle Scholar
  3. 3.
    Shen HB, Chou KC (2009) Quatldent: a web server for identifying protein quaternary structural attribute by fusing functional domain and sequential evolution information. J Proteome Res 8:1577–1584CrossRefPubMedGoogle Scholar
  4. 4.
    Otting G, Wüthrich K (1989) Extended heteronuclear editing of 2D 1H NMR spectra of isotope-labeled proteins, using the X(ω1, ω2) double half filter. J Magn Reson 85:586–594Google Scholar
  5. 5.
    Lee W, Revington MJ, Arrowsmith C et al (1994) A pulsed field gradient isotope-filtered 3D 13C HMQC-NOESY experiment for extracting intermolecular NOE contacts in molecular complexes. FEBS Lett 350:87–90CrossRefPubMedGoogle Scholar
  6. 6.
    Folmer RH, Hilbers CW, Konings RN et al (1995) A (13)C double-filtered NOESY with strongly reduced artefacts and improved sensitivity. J Biomol NMR 5:427–432CrossRefPubMedGoogle Scholar
  7. 7.
    Sobott F, Benesch JL, Vierling E et al (2002) Subunit exchange of multimeric protein complexes. Real-time monitoring of subunit exchange between small heat shock proteins by using electrospray mass spectrometry. J Biol Chem 277:38921–38929CrossRefPubMedGoogle Scholar
  8. 8.
    Pan J, Rintala-Dempsey AC, Li Y et al (2006) Folding kinetics of the S100A11 protein dimer studied by time-resolved electrospray mass spectrometry and pulsed hydrogen-deuterium exchange. Biochemistry 45:3005–3013CrossRefPubMedGoogle Scholar
  9. 9.
    Yang Y, Ramelot TA, McCarrick RM et al (2010) Combining NMR and EPR methods for homodimer protein structure determination. J Am Chem Soc 132:11910–11913CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    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 39:5355–5365CrossRefPubMedGoogle Scholar
  11. 11.
    Rumpel S, Becker S, Zweckstetter M (2008) High-resolution structure determination of the CylR2 homodimer using paramagnetic relaxation enhancement and structure-based prediction of molecular alignment. J Biomol NMR 40:1–13CrossRefPubMedGoogle Scholar
  12. 12.
    Wang X, Bansal S, Jiang M et al (2008) RDC-assisted modeling of symmetric protein homo-oligomers. Protein Sci 17:899–907CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Grishaev A, Wu J, Trewhella J et al (2005) Refinement of multidomain protein structures by combination of solution small-angle X-ray scattering and NMR data. J Am Chem Soc 127:16621–16628CrossRefPubMedGoogle Scholar
  14. 14.
    Yang Y, Ramelot TA, Cort JR et al (2011) Solution NMR structure of Dsy0195 homodimer from Desulfitobacterium hafniense: first structure representative of the YabP domain family of proteins involved in spore coat assembly. J Struct Funct Genomics 12:175–179CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Yang Y, Ramelot TA, Ni S et al (2013) Measurement of rate constants for homodimer subunit exchange using double electron–electron resonance and paramagnetic relaxation enhancements. J Biomol NMR 55:47–58CrossRefPubMedGoogle Scholar
  16. 16.
    Jeschke G, Chechik V, Ionita P et al (2006) DeerAnalysis2006—a comprehensive software package for analyzing pulsed ELDOR data. Appl Magn Reson 30:473–498CrossRefGoogle Scholar
  17. 17.
    Delaglio F, Grzesiek S, Vuister GW et al (1995) NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR 6:277–293CrossRefPubMedGoogle Scholar
  18. 18.
    Goddard TD, Kneller DG (2008) SPARKY 3. University of California, San FransiscoGoogle Scholar
  19. 19.
    Yang Y, Ramelot TA, Cort JR et al (2012) Solution NMR structure of hypothetical protein CV_2116 encoded by a viral prophage element in Chromobacterium violaceum. Int J Mol Sci 13:7354–7364CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Yang Y, Ramelot TA, Cort JR et al (2011) Solution NMR structure of photosystem II reaction center protein Psb28 from Synechocystis sp. Strain PCC 6803. Proteins 79:340–344CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Ramelot TA, Yang Y, Xiao R et al (2012) Solution NMR structure of BT_0084, a conjugative transposon lipoprotein from Bacteroides thetaiotamicron. Proteins 80:667–670CrossRefPubMedGoogle Scholar
  22. 22.
    Feldmann EA, Ramelot TA, Yang Y et al (2012) Solution NMR structure of Asl3597 from Nostoc sp. PCC7120, the first structure from protein domain family PF12095, reveals a novel fold. Proteins 80:671–675CrossRefPubMedGoogle Scholar
  23. 23.
    Bhattacharya A, Tejero R, Montelione GT (2007) Evaluating protein structures determined by structural genomics consortia. Proteins 66:778–795CrossRefPubMedGoogle Scholar
  24. 24.
    Guntert P (2004) Automated NMR structure calculation with CYANA. Methods Mol Biol 278:353–378PubMedGoogle Scholar
  25. 25.
    Linge JP, Williams MA, Spronk CA et al (2003) Refinement of protein structures in explicit solvent. Proteins 50:496–506CrossRefPubMedGoogle Scholar
  26. 26.
    Schanda P, Brutscher B (2005) Very fast two-dimensional NMR spectroscopy for real-time investigation of dynamic events in proteins on the time scale of seconds. J Am Chem Soc 127:8014–8015CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2014

Authors and Affiliations

  • Yunhuang Yang
    • 1
  • Theresa A. Ramelot
    • 1
  • Shuisong Ni
    • 2
  • Robert M. McCarrick
    • 2
  • Michael A. Kennedy
    • 1
  1. 1.Department of Chemistry and Biochemistry and the Northeast Structural Genomics ConsortiumMiami UniversityOxfordUSA
  2. 2.Department of Chemistry and BiochemistryMiami UniversityOxfordUSA

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