Skip to main content
SpringerLink
Log in

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

  • Article
  • Published:
Journal of Biomolecular NMR Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9
Fig. 10
Fig. 11
Fig. 12
Fig. 13

Similar content being viewed by others

Abbreviations

3 J C′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

References

  • 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 182:325–329

    Article  ADS  Google Scholar 

  • 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 44:3089–3092

    Article  Google Scholar 

  • 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 128:3918–3919

    Article  Google Scholar 

  • 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 53:293–301

    Article  Google Scholar 

  • 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 121:8671–8672

    Article  Google Scholar 

  • 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 20:233–249

    Article  Google Scholar 

  • 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 51:2224–2231

    Article  Google Scholar 

  • Chang SL, Tjandra N (2005) Temperature dependence of protein backbone motion from carbonyl 13C and amide 15N NMR relaxation. J Magn Reson 174:43–53

    Article  ADS  Google Scholar 

  • 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 97:213–217

    ADS  Google Scholar 

  • 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 6:277–293

    Article  Google Scholar 

  • 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 9:63–78

    Article  Google Scholar 

  • Felli IC, Pierattelli R (2014) Novel methods based on 13C detection to study intrinsically disordered proteins. J Magn Reson 241:115–125

    Article  ADS  Google Scholar 

  • 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 43:187–196

    Article  Google Scholar 

  • 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 166:39–46

    Article  ADS  Google Scholar 

  • 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 108:3246–3251

    Article  ADS  Google Scholar 

  • 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 114:6291–6293

    Article  Google Scholar 

  • Grzesiek S, Bax A (1993) Amino acid type determination in the sequential assignment procedure of uniformly 13C/15N-enriched proteins. J Biomol NMR 3:185–204

    Google Scholar 

  • Grzesiek S, Bax A (1997) A three-dimensional NMR experiment with improved sensitivity for carbonyl–carbonyl J correlation in proteins. J Biomol NMR 9:207–211

    Article  Google Scholar 

  • 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 118:8170–8171

    Article  Google Scholar 

  • 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 29:4659–4667

    Article  Google Scholar 

  • Kadkhodaie M, Rivas O, Tan M, Mohebbi A, Shaka AJ (1991) Broadband homonuclear polarization using flip–flop spectroscopy. J Magn Reson 91:437–443

    ADS  Google Scholar 

  • Kay LE, Keifer P, Saarinen T (1992) Pure absorption gradient enhanced heteronuclear single quantum correlation spectroscopy with improved sensitivity. J Am Chem Soc 114:10663–10665

    Article  Google Scholar 

  • Kay LE, Xu GY, Yamazaki T (1994) Enhanced-sensitivity triple-resonance spectroscopy with minimal H2O saturation. J Magn Reson A 109:129–133

    Article  ADS  Google Scholar 

  • Kjaergaard M, Poulsen FM (2012) Disordered proteins studied by chemical shifts. Prog Nucl Magn Reson Spectrosc 60:42–51

    Article  Google Scholar 

  • Kosol S, Contreras-Martos S, Cedeno C, Tompa P (2013) Structural characterization of intrinsically disordered proteins by NMR spectroscopy. Molecules 18:10802–10828

    Article  Google Scholar 

  • Kramer F, Peti W, Griesinger C, Glaser SJ (2001) Optimized homonuclear Carr–Purcell-type dipolar mixing sequence. J Magn Reson 149:58–66

    Article  ADS  Google Scholar 

  • 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 104:4546–4559

    Article  Google Scholar 

  • 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 104:4559–4570

    Article  Google Scholar 

  • 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 16:127–138

    Article  Google Scholar 

  • 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 3:225–231

    Article  Google Scholar 

  • 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 54:181–191

    Article  Google Scholar 

  • 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 85:393–399

    ADS  Google Scholar 

  • 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 12:1–23

    Article  Google Scholar 

  • Markwick PR, Sattler M (2004) Site-specific variations of carbonyl chemical shift anisotropies in proteins. J Am Chem Soc 126:11424–11425

    Article  Google Scholar 

  • 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 48:169–177

    Article  Google Scholar 

  • 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 61–87

    Google Scholar 

  • 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 51:199–207

    Article  Google Scholar 

  • Novacek J, Zidek L, Sklenar V (2014) Toward optimal-resolution NMR of intrinsically disordered proteins. J Magn Reson 241:41–52

    Article  ADS  Google Scholar 

  • 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 34:93–158

    Article  Google Scholar 

  • 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 123:2970–2978

    Article  Google Scholar 

  • Shaka AJ, Lee CJ, Pines A (1988) Iterative schemes for bilinear operatiors; application to spin decoupling. J Magn Reson 77:274–293

    ADS  Google Scholar 

  • Tamiola K, Mulder FAA (2012) Using NMR chemical shifts to calculate the propensity for structural order and disorder in proteins. Biochem Soc Trans 40:1014–1020

    Article  Google Scholar 

  • Tamiola K, Acar B, Mulder FAA (2010) Sequence-specific random coil chemical shifts of intrinsically disordered proteins. J Am Chem Soc 132:18000–18003

    Article  Google Scholar 

  • Uversky VN (2011) Intrinsically disordered proteins from A to Z. Int J Biochem Cell Biol 43:1090–1103

    Article  Google Scholar 

  • Uversky VN, Fink AL (2004) Conformational constraints for amyloid fibrillation: the importance of being unfolded. Biochim Biophys Acta 1698:131–153

    Article  Google Scholar 

  • Wang T, Cai S, Zuiderweg ERP (2003) Temperature dependence of anisotropic protein backbone dynamics. J Am Chem Soc 125:8639–8643

    Article  Google Scholar 

  • 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 5:67–81

    Article  Google Scholar 

  • Wright PE, Dyson HJ (1999) Intrinsically unstructured proteins: re-assessing the protein structure-function paradigm. J Mol Biol 293:321–331

    Article  Google Scholar 

  • Yao J, Dyson HJ, Wright PE (1997) Chemical shift dispersion and secondary structure prediction in unfolded and partly folded proteins. FEBS Lett 419:285–289

    Article  Google Scholar 

  • 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 241:97–102

    Article  ADS  Google Scholar 

  • Yuwen T, Skrynnikov NR (2014a) CP-HISQC: a better version of HSQC experiment for intrinsically disordered proteins under physiological conditions. J Biomol NMR 58:175–192

    Article  Google Scholar 

  • Yuwen T, Skrynnikov NR (2014b) Proton-decoupled CPMG: a better experiment for measuring 15N R2 relaxation in disordered proteins. J Magn Reson 241:155–169

    Article  ADS  Google Scholar 

Download references

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).

Author information

Authors and Affiliations

  1. Department of Chemistry and Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Gustav Wieds Vej 14, 8000, Aarhus C, Denmark

    Yuichi Yoshimura, Natalia V. Kulminskaya & Frans A. A. Mulder

Authors
  1. Yuichi Yoshimura
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Natalia V. Kulminskaya
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Frans A. A. Mulder
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Frans A. A. Mulder.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 34 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Yoshimura, Y., Kulminskaya, N.V. & Mulder, F.A.A. 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. J Biomol NMR 61, 109–121 (2015). https://doi.org/10.1007/s10858-014-9890-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10858-014-9890-7

Keywords

Search

Navigation