Advertisement

Journal of Biomolecular NMR

, Volume 61, Issue 2, pp 109–121 | Cite as

Easy and unambiguous sequential assignments of intrinsically disordered proteins by correlating the backbone 15N or 13C′ chemical shifts of multiple contiguous residues in highly resolved 3D spectra

  • Yuichi Yoshimura
  • Natalia V. Kulminskaya
  • Frans A. A. Mulder
Article

Abstract

Sequential resonance assignment strategies are typically based on matching one or two chemical shifts of adjacent residues. However, resonance overlap often leads to ambiguity in resonance assignments in particular for intrinsically disordered proteins. We investigated the potential of establishing connectivity through the three-bond couplings between sequentially adjoining backbone carbonyl carbon nuclei, combined with semi-constant time chemical shift evolution, for resonance assignments of small folded and larger unfolded proteins. Extended sequential connectivity strongly lifts chemical shift degeneracy of the backbone nuclei in disordered proteins. We show here that 3D (H)N(COCO)NH and (HN)CO(CO)NH experiments with relaxation-optimized multiple pulse mixing correlate up to seven adjacent backbone amide nitrogen or carbonyl carbon nuclei, respectively, and connections across proline residues are also obtained straightforwardly. Multiple, recurrent long-range correlations with ultra-high resolution allow backbone 1HN, 15NH, and 13C′ resonance assignments to be completed from a single pair of 3D experiments.

Keywords

Carbonyl–carbonyl J-coupling Chemical shift degeneracy Intrinsically disordered proteins Homonuclear isotropic mixing Sequential resonance assignment 

Abbreviations

3JC′C′

Three-bond coupling between sequentially adjoining 13C′ nuclei

αSyn

α-Synuclein

CSA

Chemical shift anisotropy

DSS

4,4-Dimethyl-4-silapentane-1-sulfonate

IDP

Intrinsically disordered protein

MOCCA-XY16

Modified phase cycled Carr–Purcell multiple pulse sequence with XY16 supercycles

RF

Radiofrequency

Notes

Acknowledgments

We thank Camilla B. Andersen and Tania A. Nielsen (Aarhus University, Denmark) for the expression and purification of αSyn and ubiquitin, respectively. Y.Y. is supported by an EMBO Long-Term Fellowship (ALTF 687-2013).

Supplementary material

10858_2014_9890_MOESM1_ESM.docx (34 kb)
Supplementary material 1 (DOCX 34 kb)

References

  1. Balayssac S, Jimenez B, Piccioli M (2006) 13C direct detected COCO-TOCSY: a tool for sequence specific assignment and structure determination in protonless NMR experiments. J Magn Reson 182:325–329CrossRefADSGoogle Scholar
  2. Bermel W, Bertini I, Duma L, Felli IC, Emsley L, Pierattelli R, Vasos PR (2005) Complete assignment of heteronuclear protein resonances by protonless NMR spectroscopy. Angew Chem Int Ed 44:3089–3092CrossRefGoogle Scholar
  3. Bermel W, Bertini I, Felli IC, Lee YM, Luchinat C, Pierattelli R (2006) Protonless NMR experiments for sequence-specific assignment of backbone nuclei in unfolded proteins. J Am Chem Soc 128:3918–3919CrossRefGoogle Scholar
  4. Bermel W, Bertini I, Felli IC, Gonnelli L, Kozminski W, Piai A, Pierattelli R, Stanek J (2012) Speeding up sequence specific assignment of IDPs. J Biomol NMR 53:293–301CrossRefGoogle Scholar
  5. Buevich AV, Baum J (1999) Dynamics of unfolded proteins: incorporation of distributions of correlation times in the model free analysis of NMR relaxation data. J Am Chem Soc 121:8671–8672CrossRefGoogle Scholar
  6. Buevich AV, Shinde UP, Inouye M, Baum J (2001) Backbone dynamics of the natively unfolded pro-peptide of subtilisin by heteronuclear NMR relaxation studies. J Biomol NMR 20:233–249CrossRefGoogle Scholar
  7. Camilloni C, De Simone A, Vranken WF, Vendruscolo M (2012) Determination of secondary structure populations in disordered states of proteins using nuclear magnetic resonance chemical shifts. Biochemistry 51:2224–2231CrossRefGoogle Scholar
  8. Chang SL, Tjandra N (2005) Temperature dependence of protein backbone motion from carbonyl 13C and amide 15N NMR relaxation. J Magn Reson 174:43–53CrossRefADSGoogle Scholar
  9. Clubb RT, Thanabal V, Wagner G (1992) A constant-time three-dimensional triple-resonance pulse scheme to correlate intraresidue 1HN, 15N, and 13C′ chemical shifts in 15N–13C-labeled proteins. J Magn Reson 97:213–217ADSGoogle 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. Engelke J, Ruterjans H (1997) Backbone dynamics of proteins derived from carbonyl carbon relaxation times at 500, 600 and 800 MHz: application to ribonuclease T1. J Biomol NMR 9:63–78CrossRefGoogle Scholar
  12. Felli IC, Pierattelli R (2014) Novel methods based on 13C detection to study intrinsically disordered proteins. J Magn Reson 241:115–125CrossRefADSGoogle Scholar
  13. Felli IC, Pierattelli R, Glaser SJ, Luy B (2009) Relaxation-optimised Hartmann–Hahn transfer using a specifically Tailored MOCCA-XY16 mixing sequence for carbonyl–carbonyl correlation spectroscopy in 13C direct detection NMR experiments. J Biomol NMR 43:187–196CrossRefGoogle Scholar
  14. Furrer J, Kramer F, Marino JP, Glaser SJ, Luy B (2004) Homonuclear Hartmann–Hahn transfer with reduced relaxation losses by use of the MOCCA-XY16 multiple pulse sequence. J Magn Reson 166:39–46CrossRefADSGoogle Scholar
  15. Giehm L, Svergun DI, Otzen DE, Vestergaard B (2011) Low-resolution structure of a vesicle disrupting α-synuclein oligomer that accumulates during fibrillation. Proc Natl Acad Sci U S A 108:3246–3251CrossRefADSGoogle Scholar
  16. Grzesiek S, Bax A (1992) Correlating backbone amide and side chain resonances in larger proteins by multiple relayed triple resonance NMR. J Am Chem Soc 114:6291–6293CrossRefGoogle Scholar
  17. 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
  18. Grzesiek S, Bax A (1997) A three-dimensional NMR experiment with improved sensitivity for carbonyl–carbonyl J correlation in proteins. J Biomol NMR 9:207–211CrossRefGoogle Scholar
  19. Hu JS, Bax A (1996) Measurement of three bond 13C–13C J couplings between carbonyl and carbonyl/carboxyl carbons in isotopically enriched proteins. J Am Chem Soc 118:8170–8171CrossRefGoogle Scholar
  20. Ikura M, Kay LE, Bax A (1990) A novel approach for sequential assignment of proton, carbon-13, and nitrogen-15 spectra of larger proteins: heteronuclear triple-resonance three-dimensional NMR spectroscopy. Application to calmodulin. Biochemistry 29:4659–4667CrossRefGoogle Scholar
  21. Kadkhodaie M, Rivas O, Tan M, Mohebbi A, Shaka AJ (1991) Broadband homonuclear polarization using flip–flop spectroscopy. J Magn Reson 91:437–443ADSGoogle Scholar
  22. Kay LE, Keifer P, Saarinen T (1992) Pure absorption gradient enhanced heteronuclear single quantum correlation spectroscopy with improved sensitivity. J Am Chem Soc 114:10663–10665CrossRefGoogle Scholar
  23. Kay LE, Xu GY, Yamazaki T (1994) Enhanced-sensitivity triple-resonance spectroscopy with minimal H2O saturation. J Magn Reson A 109:129–133CrossRefADSGoogle Scholar
  24. Kjaergaard M, Poulsen FM (2012) Disordered proteins studied by chemical shifts. Prog Nucl Magn Reson Spectrosc 60:42–51CrossRefGoogle Scholar
  25. Kosol S, Contreras-Martos S, Cedeno C, Tompa P (2013) Structural characterization of intrinsically disordered proteins by NMR spectroscopy. Molecules 18:10802–10828CrossRefGoogle Scholar
  26. Kramer F, Peti W, Griesinger C, Glaser SJ (2001) Optimized homonuclear Carr–Purcell-type dipolar mixing sequence. J Magn Reson 149:58–66CrossRefADSGoogle Scholar
  27. Lipari G, Szabo A (1982a) 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
  28. Lipari G, Szabo A (1982b) Model-free approach to the interpretation of nuclear magnetic resonance relaxation in macromolecules. 2. Analysis of experimental results. J Am Chem Soc 104:4559–4570CrossRefGoogle Scholar
  29. Liu A, Riek R, Wider G, von Schroetter C, Zahn R, Wüthrich K (2000) NMR experiments for resonance assignments of 13C, 15N doubly-labeled flexible polypeptides: application to the human prion protein hPrP(23–230). J Biomol NMR 16:127–138CrossRefGoogle Scholar
  30. Logan TM, Olejniczak ET, Xu RX, Fesik SW (1993) A general method for assigning NMR spectra of denatured proteins using 3D HC(CO)NH-TOCSY triple resonance experiments. J Biomol NMR 3:225–231CrossRefGoogle Scholar
  31. Maltsev AS, Ying J, Bax A (2012) Deuterium isotope shifts for backbone 1H, 15N and 13C nuclei in intrinsically disordered protein and α-synuclein. J Biomol NMR 54:181–191CrossRefGoogle Scholar
  32. Marion D, Ikura M, Tschudin R, Bax A (1989) Rapid recording of 2D NMR spectra without phase cycling. Application to the study of hydrogen exchange in proteins. J Magn Reson 85:393–399ADSGoogle Scholar
  33. Markley JL, Bax A, Arata Y, Hilbers CW, Kaptein R, Sykes BD, Wright PE, Wuthrich K (1998) Recommendations for the presentation of NMR structures of proteins and nucleic acids. IUPAC–IUBMB–IUPAB inter-union task group on the standardization of data bases of protein and nucleic acid structures determined by NMR spectroscopy. J Biomol NMR 12:1–23CrossRefGoogle Scholar
  34. Markwick PR, Sattler M (2004) Site-specific variations of carbonyl chemical shift anisotropies in proteins. J Am Chem Soc 126:11424–11425CrossRefGoogle Scholar
  35. Motackova V, Novacek J, Zawadzka-Kazimierczuk A, Kazimierczuk K, Zidek L, Sanderova H, Krasny L, Kozminski W, Sklenar V (2010) Strategy for complete NMR assignment of disordered proteins with highly repetitive sequences based on resolution-enhanced 5D experiments. J Biomol NMR 48:169–177CrossRefGoogle Scholar
  36. Mulder FAA, Lundqvist M, Scheek RM (2010) Nuclear magnetic resonance spectroscopy applied to (intrinsically) disordered proteinsin. In: Uversky VN, Longhi S (eds) Instrumental analysis of intrinsically disordered proteins: assessing structure and conformation. Wiley, Hoboken, pp 61–87Google Scholar
  37. Mulder FAA, Otten R, Scheek RM (2011) Origin and removal of mixed-phase artifacts in gradient sensitivity enhanced heteronuclear single quantum correlation spectra. J Biomol NMR 51:199–207CrossRefGoogle Scholar
  38. Novacek J, Zidek L, Sklenar V (2014) Toward optimal-resolution NMR of intrinsically disordered proteins. J Magn Reson 241:41–52CrossRefADSGoogle Scholar
  39. Sattler M, Schleucher J, Griesinger C (1999) Heteronuclear multidimensional NMR experiments for the structure determination of proteins in solution employing pulsed field gradients. Prog Nucl Magn Reson Spectrosc 34:93–158CrossRefGoogle Scholar
  40. Schwarzinger S, Kroon GJ, Foss TR, Chung J, Wright PE, Dyson HJ (2001) Sequence-dependent correction of random coil NMR chemical shifts. J Am Chem Soc 123:2970–2978CrossRefGoogle Scholar
  41. Shaka AJ, Lee CJ, Pines A (1988) Iterative schemes for bilinear operatiors; application to spin decoupling. J Magn Reson 77:274–293ADSGoogle Scholar
  42. Tamiola K, Mulder FAA (2012) Using NMR chemical shifts to calculate the propensity for structural order and disorder in proteins. Biochem Soc Trans 40:1014–1020CrossRefGoogle Scholar
  43. Tamiola K, Acar B, Mulder FAA (2010) Sequence-specific random coil chemical shifts of intrinsically disordered proteins. J Am Chem Soc 132:18000–18003CrossRefGoogle Scholar
  44. Uversky VN (2011) Intrinsically disordered proteins from A to Z. Int J Biochem Cell Biol 43:1090–1103CrossRefGoogle Scholar
  45. Uversky VN, Fink AL (2004) Conformational constraints for amyloid fibrillation: the importance of being unfolded. Biochim Biophys Acta 1698:131–153CrossRefGoogle Scholar
  46. Wang T, Cai S, Zuiderweg ERP (2003) Temperature dependence of anisotropic protein backbone dynamics. J Am Chem Soc 125:8639–8643CrossRefGoogle Scholar
  47. Wishart DS, Bigam CG, Holm A, Hodges RS, Sykes BD (1995) 1H, 13C and 15N random coil NMR chemical shifts of the common amino acids. I. Investigations of nearest-neighbor effects. J Biomol NMR 5:67–81CrossRefGoogle Scholar
  48. Wright PE, Dyson HJ (1999) Intrinsically unstructured proteins: re-assessing the protein structure-function paradigm. J Mol Biol 293:321–331CrossRefGoogle Scholar
  49. Yao J, Dyson HJ, Wright PE (1997) Chemical shift dispersion and secondary structure prediction in unfolded and partly folded proteins. FEBS Lett 419:285–289CrossRefGoogle Scholar
  50. Ying J, Roche J, Bax A (2014) Homonuclear decoupling for enhancing resolution and sensitivity in NOE and RDC measurements of peptides and proteins. J Magn Reson 241:97–102CrossRefADSGoogle Scholar
  51. Yuwen T, Skrynnikov NR (2014a) CP-HISQC: a better version of HSQC experiment for intrinsically disordered proteins under physiological conditions. J Biomol NMR 58:175–192CrossRefGoogle Scholar
  52. Yuwen T, Skrynnikov NR (2014b) Proton-decoupled CPMG: a better experiment for measuring 15N R2 relaxation in disordered proteins. J Magn Reson 241:155–169CrossRefADSGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2015

Authors and Affiliations

  • Yuichi Yoshimura
    • 1
  • Natalia V. Kulminskaya
    • 1
  • Frans A. A. Mulder
    • 1
  1. 1.Department of Chemistry and Interdisciplinary Nanoscience Center (iNANO)Aarhus UniversityAarhus CDenmark

Personalised recommendations