Skip to main content
SpringerLink
Log in

“CON-CON” assignment strategy for highly flexible intrinsically disordered proteins

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

Abstract

Intrinsically disordered proteins (IDPs) are a class of highly flexible proteins whose characterization by NMR spectroscopy is complicated by severe spectral overlaps. The development of experiments designed to facilitate the sequence-specific assignment procedure is thus very important to improve the tools for the characterization of IDPs and thus to be able to focus on IDPs of increasing size and complexity. Here, we present and describe the implementation of a set of novel 1H-detected 5D experiments, (HACA)CON(CACO)NCO(CA)HA, BT-(H)NCO(CAN)CONNH and BT-HN(COCAN)CONNH, optimized for the study of highly flexible IDPs that exploit the best resolved correlations, those involving the carbonyl and nitrogen nuclei of neighboring amino acids, to achieve sequence-specific resonance assignment. Together with the analogous recently proposed pulse schemes based on 13C detection, they form a complete set of experiments for sequence-specific assignment of highly flexible IDPs. Depending on the particular sample conditions (concentration, lifetime, pH, temperature, etc.), these experiments present certain advantages and disadvantages that will be discussed. Needless to say, that the availability of a variety of complementary experiments will be important for accurate determination of resonance frequencies in complex IDPs.

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

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

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

Similar content being viewed by others

References

  • Bermel W, Bertini I, Gonnelli L, Felli IC, Koźmiński W, Piai A, Pierattelli R, Stanek J (2012) Speeding up sequence specific assignment of IDPs. J Biomol NMR 53:293–301

    Article  Google Scholar 

  • Bermel W, Bruix M, Felli IC, Kumar VMV, Pierattelli R, Serrano S (2013a) Improving the chemical shift dispersion of multidimensional NMR spectra of intrinsically disordered proteins. J Biomol NMR 55:231–237

    Article  Google Scholar 

  • Bermel W, Felli IC, Gonnelli L, Koźmiński W, Piai A, Pierattelli R, Zawadzka-Kazimierczuk A (2013b) High-dimensionality 13C direct-detected NMR experiments for the automatic assignment of intrinsically disordered proteins. J Biomol NMR 57:353–361

    Article  Google Scholar 

  • Böhlen J-M, Bodenhausen G (1993) Experimental aspects of chirp NMR spectroscopy. J Magn Reson Ser A 102:293–301

    Article  ADS  Google Scholar 

  • Cavanagh J, Rance M (1992) Suppression of cross-relaxation effects in TOCSY spectra via a modified DIPSI-2 mixing sequence. J Magn Reson 96:670–678

    ADS  Google Scholar 

  • Csizmok V, Felli IC, Tompa P, Banci L, Bertini I (2008) Structural and dynamic characterization of intrinsically disordered human securin by NMR. J Am Chem Soc 130:16873–16879

    Article  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 

  • Deschamps M, Campbell ID (2006) Cooling overall spin temperature: protein NMR experiments optimized for longitudinal relaxation effects. J Magn Reson 178:206–211

    Article  ADS  Google Scholar 

  • Dunker AK, Lawson JD, Brown CJ, Williams RM, Romero P, Oh JS, Ratliff CM, Hipps KW, Ausio J, Nissen MS, Reeves R, Kang C, Kissinger CR, Bailey RW, Griswold MD, Chiu W, Garner EC (2001) Intrinsically disordered protein. J Mol Graph Model 19:26–59

    Article  Google Scholar 

  • Dyson HJ, Wright PE (2004) Unfolded proteins and protein folding studied by NMR. Chem Rev 104:3607–3622

    Article  Google Scholar 

  • Emsley L, Bodenhausen G (1990) Gaussian pulse cascades: new analytical functions for rectangular selective inversion and in-phase excitation in NMR. Chem Phys Lett 165:469–476

    Article  ADS  Google Scholar 

  • Emsley L, Bodenhausen G (1992) Optimization of shaped selective pulses for NMR using a quaternion description of their overall propagators. J Magn Reson 97:135–148

    ADS  Google Scholar 

  • Favier A, Brutscher B (2011) Recovering lost magnetization: polarization enhancement in biomolecular NMR. J Biomol NMR 49:9–15

    Article  Google Scholar 

  • Felli IC, Brutscher B (2009) Recent advancements in solution NMR: fast methods and heteronuclear direct detection. ChemPhysChem 10:1356–1368

    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, Piai A, Pierattelli R (2013) Recent advances in solution NMR studies: 13C direct detection for biomolecular NMR applications. Ann Rep NMR Spectroscop 80:359–418

  • Geen H, Freeman R (1991) Band-selective radiofrequency pulses. J Magn Reson 93:93–141

    ADS  Google Scholar 

  • Gil S, Hošek T, Solyom Z, Kümmerle R, Brutscher B, Pierattelli R, Felli IC (2013) NMR studies of intrinsically disordered proteins near physiological conditions. Angew Chem Int Ed 52:11808–11812

    Article  Google Scholar 

  • Goddard TD, Kneller DG (2000) SPARKY 3. University of California, San Francisco

    Google Scholar 

  • Hiller S, Wasmer C, Wider G, Wüthrich K (2007) Sequence-specific resonance assignment of soluble nonglobular proteins by 7D APSY-NMR spectroscopy. J Am Chem Soc 129:10823–10828

    Article  Google Scholar 

  • Hsu ST, Bertoncini CW, Dobson CM (2009) Use of protonless NMR spectroscopy to alleviate the loss of information resulting from exchange-broadening. J Am Chem Soc 131:7222–7223

    Article  Google Scholar 

  • Huang C, Ren G, Zhou H, Wang C (2005) A new method for purification of recombinant human alpha-synuclein in Escherichia coli. Protein Expr Purif 42:173–177

    Article  Google Scholar 

  • Ikura M, Spera S, Barbato G, Kay LE, Krinks M, Bax A (1991) Secondary structure and side-chain 1H and 13C resonance assignments of calmodulin in solution by heteronuclear multidimensional NMR spectroscopy. Biochemistry 30:9216–9228

    Article  Google Scholar 

  • Kazimierczuk K, Zawadzka A, Koźmiński W, Zhukov I (2006) Random sampling of evolution time space and Fourier transform processing. J Biomol NMR 36:157–168

    Article  Google Scholar 

  • Kazimierczuk K, Zawadzka A, Koźmiński W (2008) Optimization of random time domain sampling in multidimensional NMR. J Magn Reson 192:123–130

    Article  ADS  Google Scholar 

  • Kazimierczuk K, Zawadzka A, Koźmiński W (2009) Narrow peaks and high dimensionalities: exploiting the advantages of random sampling. J Magn Reson 197:219–228

    Article  ADS  Google Scholar 

  • Kazimierczuk K, Stanek J, Zawadzka-Kazimierczuk A, Koźmiński W (2010a) Random sampling in multidimensional NMR spectroscopy. Prog NMR Spectrosc 57:420–434

    Article  Google Scholar 

  • Kazimierczuk K, Zawadzka-Kazimierczuk A, Koźmiński W (2010b) Non-uniform frequency domain for optimal exploitation of non-uniform sampling. J Magn Reson 205:286–292

    Article  ADS  Google Scholar 

  • Kazimierczuk K, Misiak M, Stanek J, Zawadzka-Kazimierczuk A, Koźmiński W (2012) Generalized Fourier transform for non-uniform sampled data. Top Curr Chem 316:79–124

    Article  Google Scholar 

  • Kazimierczuk K, Stanek J, Zawadzka-Kazimierczuk A, Koźmiński W (2013) High-dimensional NMR spectra for structural studies of biomolecules. ChemPhysChem 14:3015–3025

    Article  Google Scholar 

  • Knoblich K, Whittaker S, Ludwig C, Michiels P, Jiang T, Schafflhausen B, Günther U (2009) Backbone assignment of the N-terminal polyomavirus large T antigen. Biomol NMR Assign 3:119–123

    Article  Google Scholar 

  • Konrat R (2014) NMR contributions to structural dynamics studies of intrinsically disordered proteins. J Magn Reson 241:74–85

    Article  ADS  Google Scholar 

  • Kupce E, Freeman R (2003) Projection-reconstruction of three-dimensional NMR spectra. J Am Chem Soc 125:13958–13959

    Article  Google Scholar 

  • Mäntylahti S, Aitio O, Hellman M, Permi P (2010) HA-detected experiments for the backbone assignment of intrinsically disordered proteins. J Biomol NMR 47:171–181

    Article  Google Scholar 

  • Mäntylahti S, Hellman M, Permi P (2011) Extension of the HA-detection based approach: (HCA)CON(CA)H and (HCA)NCO(CA)H experiments for the main-chain assignment of intrinsically disordered proteins. J Biomol NMR 49:99–109

    Article  Google Scholar 

  • Mittag T, Forman-Kay J (2007) Atomic-level characterization of disordered protein ensembles. Curr Opin Struct Biol 17:3–14

    Article  Google Scholar 

  • Motáčkova V, Nováček J, Zawadzka-Kazimierczuk A, Kazimierczuk K, Židek L, Sanderová H, Krásný L, Koźmiński W, Sklenář 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 

  • Narayanan RL, Dürr HN, Bilbow S, Biernat J, Mendelkow E, Zweckstetter M (2010) Automatic assignment of the intrinsically disordered protein Tau with 441-residues. J Am Chem Soc 132:11906–11907

    Article  Google Scholar 

  • Nováček J, Zawadzka-Kazimierczuk A, Papoušková V, Židek L, Sanderová H, Krásný L, Koźmiński W, Sklenář V (2011) 5D 13C-detected experiments for backbone assignment of unstructured proteins with a very low signal dispersion. J Biomol NMR 50:1–11

    Article  Google Scholar 

  • Nováček J, Janda L, Dopitová R, Židek L, Sklenář V (2013) Efficient protocol for backbone and side-chain assignments of large, intrinsically disordered proteins: transient secondary structure analysis of 49.2 kDa microtubule associated protein 2c. J Biomol NMR 56:291–301

    Article  Google Scholar 

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

    Article  ADS  Google Scholar 

  • O’Hare B, Benesi AJ, Showalter SA (2009) Incorporating 1H chemical shift determination into 13C-direct detected spectroscopy of intrinsically disordered proteins in solution. J Magn Reson 200:354–358

    Article  ADS  Google Scholar 

  • Panchal SC, Bhavesh NS, Hosur RV (2001) Improved 3D triple resonance experiments, HNN and HN(C)N, for HN and 15N sequential correlations (13C, 15N) labeled proteins: application to unfolded proteins. J Biomol NMR 20:135–147

    Article  Google Scholar 

  • Pantoja-Uceda D, Santoro J (2013a) A suite of amino acid residue type classification pulse sequences for 13C-detected NMR of proteins. J Magn Reson 234:190–196

    Article  ADS  Google Scholar 

  • Pantoja-Uceda D, Santoro J (2013b) Direct correlation of consecutive C′–N groups in proteins: a method for the assignment of intrinsically disordered proteins. J Biomol NMR 57:57–63

    Article  Google Scholar 

  • Pantoja-Uceda D, Santoro J (2014) New 13C-detected experiments for the assignment of intrinsically disordered proteins. J Biomol NMR 59:43–50

    Article  Google Scholar 

  • Pérez Y, Gairí M, Pons M, Bernadò P (2009) Structural characterization of the natively unfolded N-terminal domain of human c-Src kinase: insights into the role of phosphorylation of the unique domain. J Mol Biol 391:136–148

    Article  Google Scholar 

  • Pervushin K, Riek R, Wider G, Wüthrich K (1997) Attenuated T-2 relaxation by mutual cancellation of dipole-dipole coupling and chemical shift anisotropy indicates an avenue to NMR structures of very large biological macromolecules in solution. Proc Natl Acad Sci USA 94:12366–12371

    Article  ADS  Google Scholar 

  • Pervushin K, Vogeli B, Eletsky A (2002) Longitudinal (1)H relaxation optimization in TROSY NMR spectroscopy. J Am Chem Soc 124:12898–12902

    Article  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. Progr NMR Spectrosc 34:93–158

    Article  Google Scholar 

  • Schanda P (2009) Fast-pulsing longitudinal relaxation optimized techniques: enriching the toolbox. Prog NMR Spectrosc 55:238–265

    Article  Google Scholar 

  • 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 127:8014–8015

    Article  Google Scholar 

  • Schanda P, Kupce E, Brutscher B (2005) SOFAST-HMQC experiments for recording two-dimensional heteronuclear correlation spectra of proteins within a few seconds. J Biomol NMR 33:199–211

    Article  Google Scholar 

  • Schanda P, Van Melckebeke H, Brutscher B (2006) Speeding up three-dimensional protein NMR experiments to a few minutes. J Am Chem Soc 128:9042–9043

    Article  Google Scholar 

  • Shaka AJ, Barker PB, Freeman R (1985) Computer-optimized decoupling scheme for wideband applications and low-level operation. J Magn Reson 64:547–552

    ADS  Google Scholar 

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

    ADS  Google Scholar 

  • Skora L, Becker S, Zweckstetter M (2010) Molten globule precursor states are conformationally correlated to amyloid fibrils of human beta-2-microglobulin. J Am Chem Soc 132:9223–9225

    Article  Google Scholar 

  • Smith MA, Hu H, Shaka AJ (2001) Improved broadband inversion performance for NMR in liquids. J Magn Reson 151:269–283

    Article  ADS  Google Scholar 

  • Solyom Z, Schwarten M, Geist L, Konrat R, Willbold D, Brutscher B (2013) BEST-TROSY experiments for time-efficient sequential resonance assignment of large disordered proteins. J Biomol NMR 55:311–321

    Article  Google Scholar 

  • Stanek J, Augustyniak R, Koźmiński W (2012) Suppression of sampling artefacts in high-resolution four-dimensional NMR spectra using signal separation algorithm. J Magn Reson 214:91–102

    Article  ADS  Google Scholar 

  • Tompa P (2009) Structure and function of intrinsically disordered proteins. CRC Press, Boca Raton

    Book  Google Scholar 

  • Tompa P (2012) Intrinsically disordered proteins: a 10-year recap. Trends Biochem Sci 37:509–516

    Article  Google Scholar 

  • Uversky VN (2013a) A decade and a half of protein intrinsic disorder: biology still waits for physics. Protein Sci 22:693–724

    Article  Google Scholar 

  • Uversky VN (2013b) Multitude of binding modes attainable by intrinsically disordered proteins: a portrait gallery of disorder-based complexes. Chem Soc Rev 40:1623–1634

    Article  Google Scholar 

  • Uversky V, Dunker AK (2013) The case for intrinsically disordered proteins playing contributory roles in molecular recognition without a stable 3D structure. F1000 Biol Rep 5:1

    Article  Google Scholar 

  • Uversky VN, Gillespie JR, Fink AL (2000) Why are “natively unfolded” proteins unstructured under physiologic conditions? Proteins Struct Funct Genet 41:415–427

    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 

  • Zawadzka-Kazimierczuk A, Koźmiński W, Billeter M (2012a) TSAR: a program for automatic resonance assignment using 2D cross-sections of high dimensionality, high-resolution spectra. J Biomol NMR 54:81–95

    Article  Google Scholar 

  • Zawadzka-Kazimierczuk A, Koźmiński W, Sanderová H, Krásný L (2012b) High dimensional and high resolution pulse sequences for backbone resonance assignment of intrinsically disordered proteins. J Biomol NMR 52:329–337

    Article  Google Scholar 

Download references

Acknowledgments

This work has been supported in part by the European Commission Projects IDPbyNMR (Contract No. 264257), BioNMR (Contract No. 261863) and INSTRUCT (Contract No. 211252).

Author information

Authors and Affiliations

  1. CERM and Department of Chemistry, University of Florence, Via Luigi Sacconi 6, 50019, Sesto Fiorentino, Florence, Italy

    Alessandro Piai, Tomáš Hošek, Leonardo Gonnelli, Roberta Pierattelli & Isabella C. Felli

  2. Faculty of Chemistry, Biological and Chemical Research Centre, University of Warsaw, Żwirki i Wigury 101, 02-089, Warsaw, Poland

    Anna Zawadzka-Kazimierczuk & Wiktor Koźmiński

  3. Institut de Biologie Structurale, Université Grenoble 1, CNRS, CEA, Rue Jules Horowitz 41, 38027, Grenoble Cedex 1, France

    Bernhard Brutscher

  4. Bruker BioSpin GmbH, Silberstreifen, 76287, Rheinstetten, Germany

    Wolfgang Bermel

Authors
  1. Alessandro Piai
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tomáš Hošek
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Leonardo Gonnelli
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Anna Zawadzka-Kazimierczuk
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Wiktor Koźmiński
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Bernhard Brutscher
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Wolfgang Bermel
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Roberta Pierattelli
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Isabella C. Felli
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Isabella C. Felli.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 708 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Piai, A., Hošek, T., Gonnelli, L. et al. “CON-CON” assignment strategy for highly flexible intrinsically disordered proteins. J Biomol NMR 60, 209–218 (2014). https://doi.org/10.1007/s10858-014-9867-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10858-014-9867-6

Keywords

Search

Navigation