Journal of Biomolecular NMR

, Volume 37, Issue 3, pp 179–185 | Cite as

Pairwise NMR experiments for the determination of protein backbone dihedral angle Φ based on cross-correlated spin relaxation

  • Hideo TakahashiEmail author
  • Ichio ShimadaEmail author
Original paper


Novel cross-correlated spin relaxation (CCR) experiments are described, which measure pairwise CCR rates for obtaining peptide dihedral angles Φ. The experiments utilize intra-HNCA type coherence transfer to refocus 2-bond \({J_{{\rm NC}\alpha}}\) coupling evolution and generate the \({\hbox{N}(i)\hbox{-\!-}\hbox{C}^{\alpha}(i)}\) or \({\hbox{C}^{\prime}(i-1)\hbox{-\!-}\hbox{C}^{\alpha}(i)}\) multiple quantum coherences which are required for measuring the desired CCR rates. The contribution from other coherences is also discussed and an appropriate setting of the evolution delays is presented. These CCR experiments were applied to 15N- and 13C-labeled human ubiquitin. The relevant CCR rates showed a high degree of correlation with the Φ angles observed in the X-ray structure. By utilizing these CCR experiments in combination with those previously established for obtaining dihedral angle Ψ, we can determine high resolution structures of peptides that bind weakly to large target molecules.


Chemical shift anisotropy Cross-correlated relaxation Dihedral angle Dipole–dipole interaction Intra-HNCA Structure determination 


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This work was supported by grants from the New Energy and Industrial Technology Development Organization.


  1. Blommers MJJ, Stark W, Jones CE, Head D, Owen CE Jahnke W (1999) Transferred cross-correlated relaxation complements transferred NOE: structure of an IL-4R-derived peptide bound to STAT-6. J Am Chem Soc 121:1949–1953CrossRefGoogle Scholar
  2. Bohlen JM, Bodenhausen G (1993) Experimental aspects of chirp NMR-spectroscopy. J Magn Reson A 102:293–301CrossRefGoogle Scholar
  3. Brutscher B (2002) Intraresidue HNCA and COHNCA experiments for protein backbone resonance assignment. J Magn Reson 156:155–159CrossRefADSGoogle Scholar
  4. Carlomagno T, Griesinger C (2000) Errors in the measurement of cross-correlated relaxation rates and how to avoid them. J␣Magn Reson 144:280–287CrossRefADSGoogle Scholar
  5. Carlomagno T, Felli IC, Czech M, Fischer R, Sprinzl M, Griesinger C (1999) Transferred cross-correlated relaxation: application to the determination of sugar pucker in an aminoacylated tRNA-mimetic weakly bound to EF-Tu. J Am Chem Soc 121:1945–1948CrossRefGoogle Scholar
  6. Carlomagno T, Blommers MJJ, Meiler J, Jahnke W, Schupp T, Petersen F, Schinzer D, Altmann KH, Griesinger C (2003a) The high-resolution solution structure of epothilone A bound to tubulin: an understanding of the structure-activity relationships for a powerful class of antitumor agents. Angew Chem Int Ed Engl 42:2511–2515CrossRefGoogle Scholar
  7. Carlomagno T, Sánchez VM, Blommers MJJ, Griesinger C (2003b) Derivation of dihedral angles from CH–CH dipolar-dipolar cross-correlated relaxation rates: A C–C torsion involving a quaternary carbon atom in epothilone A bound to tubulin. Angew Chem Int Ed Engl 42:2515–2517CrossRefGoogle Scholar
  8. Chiarparin E, Pelupessy P, Ghose R, Bodenhausen G (1999) Relaxation of two-spin coherence due to cross-correlated fluctuations of dipole-dipole couplings and anisotropic shifts in NMR of 15N, 13C-labeled biomolecules. J Am Chem Soc 121: 6876–6883CrossRefGoogle Scholar
  9. Chiarparin E, Pelupessy P, Ghose R, Bodenhausen G (2000) Relative orientation of CαHα-bond vectors of successive residues in proteins through cross-correlated relaxation in NMR. J Am Chem Soc 122:1758–1761CrossRefGoogle Scholar
  10. Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, Bax A (1995) Nmrpipe – a multidimensional spectral processing system based on unix pipes. J Biomol NMR 6:277–293CrossRefGoogle Scholar
  11. Delaglio F, Torchia DA, Bax A (1991) Measurement of 15N–13C J couplings in staphylococcal nuclease. J Biomol NMR 1:439–466CrossRefGoogle Scholar
  12. Grzesiek S, Bax A (1993) Amino acid type determination in the␣sequential assignment procedure of uniformly 13C/15N-enriched proteins. J Biomol NMR 3:185–204Google Scholar
  13. Kloiber K, Konrat R (2000) Measurement of the protein backbone dihedral angle ϕ based on quantification of␣remote CSA/DD interference in inter-residue 13C’(i-1)–13Cα(i) multiple-quantum coherences. J Biomol NMR 17:265–268CrossRefGoogle Scholar
  14. Kloiber K, Schuler W, Konrat R (2002) Automated NMR determination of protein backbone dihedral angles from cross-correlated spin relaxation. J Biomol NMR 22:349–363CrossRefGoogle Scholar
  15. Nietlispach D, Ito Y, Laue ED (2002) A novel approach for the sequential backbone assignment of larger proteins: Selective intra-HNCA and DQ-HNCA. J Am Chem Soc 124:11199–11207CrossRefGoogle Scholar
  16. Pelupessy P, Chiarparin E, Ghose R, Bodenhausen G (1999a) Efficient determination of angles subtended by Cα–Hα and N–HN vectors in proteins via dipole-dipole cross-correlation. J Biomol NMR 13:375–380CrossRefGoogle Scholar
  17. Pelupessy P, Chiarparin E, Ghose R, Bodenhausen G (1999b) Simultaneous determination of Ψ and Φ angles in proteins from measurements of cross-correlated relaxation effects. J␣Biomol NMR 14:277–280CrossRefGoogle Scholar
  18. Permi P (2002) Intraresidual HNCA: An experiment for correlating only intraresidual backbone resonances. J Biomol NMR 23:201–209CrossRefGoogle Scholar
  19. Reif B, Hennig M, Griesinger C (1997) Direct measurement of angles between bond vectors in high-resolution NMR. Science 276:1230–1233CrossRefGoogle Scholar
  20. Sklenar V, Piotto M, Leppik R, Saudek V (1993) Gradient-tailored water suppression for 1H–15N HSQC experiments optimized to retain full sensitivity. J Magn Reson A␣102:241–245CrossRefGoogle Scholar
  21. Sprangers R, Bottomley MJ, Linge JP, Schultz J, Nilges M, Sattler M (2000) Refinement of the protein backbone angle ψ in NMR structure calculations. J Biomol NMR 16:47–58CrossRefGoogle Scholar
  22. Teng Q, Iqbal M, Cross TA (1992) Determination of the 13C chemical-shift and 14N electric field gradient tensor orientations with respect to the molecular frame in a polypeptide. J Am Chem Soc 114:5312–5321CrossRefGoogle Scholar
  23. Tjandra N, Feller SE, Pastor RW, Bax A (1995) Rotational diffusion anisotropy of human ubiquitin from 15N NMR relaxation. J Am Chem Soc 117:12562–12566CrossRefGoogle Scholar
  24. Wirmer J, Schwalbe H (2002) Angular dependence of 1J(Ni,Cαi) and 2J(Ni,Cα(i−1)) coupling constants measured in J-modulated HSQCs. J Biomol NMR 23:47–55CrossRefGoogle Scholar
  25. Yang DW, Kay LE (1998) Determination of the protein backbone dihedral angle ψ from a combination of NMR-derived cross-correlation spin relaxation rates. J Am Chem Soc 120:9880–9887CrossRefGoogle Scholar
  26. Yang DW, Konrat R, Kay LE (1997) A multidimensional NMR experiment for measurement of the protein dihedral angle ψ based on cross-correlated relaxation between 1Hα13Cα dipolar and 13C’ (carbonyl) chemical shift anisotropy mechanisms. J Am Chem Soc 119:11938–11940CrossRefGoogle Scholar
  27. Yang DW, Gardner KH, Kay LE (1998) A sensitive pulse scheme for measuring the backbone dihedral angle ψ based on cross-correlation between 13Cα1Hα dipolar and carbonyl chemical shift anisotropy relaxation interactions. J Biomol NMR 11:213–220CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2007

Authors and Affiliations

  1. 1.Biological Information Research Center (BIRC)National Institute of Advanced Industrial Science and Technology (AIST)TokyoJapan
  2. 2.Graduate School of Pharmaceutical SciencesThe University of TokyoTokyoJapan

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