Journal of Biomolecular NMR

, Volume 70, Issue 3, pp 167–175 | Cite as

13C APSY-NMR for sequential assignment of intrinsically disordered proteins

  • Maria Grazia Murrali
  • Marco Schiavina
  • Valerio Sainati
  • Wolfgang Bermel
  • Roberta Pierattelli
  • Isabella C. Felli


The increasingly recognized biological relevance of intrinsically disordered proteins requires a continuous expansion of the tools for their characterization via NMR spectroscopy, the only technique so far able to provide atomic-resolution information on these highly mobile macromolecules. Here we present the implementation of projection spectroscopy in 13C-direct detected NMR experiments to achieve the sequence specific assignment of IDPs. The approach was used to obtain the complete backbone assignment at high temperature of α-synuclein, a paradigmatic intrinsically disordered protein.


Intrinsically disordered proteins IDPs Assignment NUS 13C detection 



The support and the use of resources of the CERM/CIRMMP center of Instruct-ERIC, a Landmark ESFRI project, is gratefully acknowledged. This work has been supported in part by a grant of the Fondazione CR di Firenze and by MEDINTECH (CTN01_001177_962865). MAECI is also gratefully acknowledged.

Supplementary material

10858_2018_167_MOESM1_ESM.docx (308 kb)
Supplementary material 1 (DOCX 308 KB)


  1. Ambadipudi S, Zweckstetter M (2016) Targeting intrinsically disordered proteins in rational drug discovery. Expert Opin Drug Discov 11:65–77CrossRefGoogle Scholar
  2. Baias M, Smith PE, Shen K, Joachimiak LA, Zerko S, Koźmiński W, Frydman J, Frydman L (2017) Structure and dynamics of the Huntingtin exon-1 N-terminus: a solution NMR perspective. J Am Chem Soc 139:1168–1176CrossRefGoogle Scholar
  3. Bermel W, Bertini I, Felli IC, Kümmerle R, Pierattelli R (2006a) Novel 13C direct detection experiments, including extension to the third dimension, to perform the complete assignment of proteins. J Magn Reson 178:56–64ADSCrossRefGoogle Scholar
  4. Bermel W, Bertini I, Felli IC, Lee Y-M, Luchinat C, Pierattelli R (2006b) Protonless NMR experiments for sequence-specific assignment of backbone nuclei in unfolded proteins. J Am Chem Soc 128:3918–3919CrossRefGoogle Scholar
  5. Bermel W, Bertini I, Felli IC, Piccioli M, Pierattelli R (2006c) 13C-detected protonless NMR spectroscopy of proteins in solution. Progr NMR Spectrosc 48:25–45CrossRefGoogle Scholar
  6. Bermel W, Felli IC, Kümmerle R, Pierattelli R (2008) 13C direct-detection biomolecular NMR. Concepts Magn Reson 32A:183–200CrossRefGoogle Scholar
  7. Bermel W, Bertini I, Csizmok V, Felli IC, Pierattelli R, Tompa P (2009a) H-start for exclusively heteronuclear NMR spectroscopy: the case of intrinsically disordered proteins. J Magn Reson 198:275–281ADSCrossRefGoogle Scholar
  8. Bermel W, Bertini I, Felli IC, Pierattelli R (2009b) Speeding up 13C direct detection biomolecular NMR experiments. J Am Chem Soc 131:15339–15345CrossRefGoogle Scholar
  9. 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 53:293–301CrossRefGoogle Scholar
  10. 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 55:231–237CrossRefGoogle Scholar
  11. 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 57:353–361CrossRefGoogle Scholar
  12. Böhlen J-M, Bodenhausen G (1993) Experimental aspects of chirp NMR spectroscopy. J Magn Reson Ser A 102:293–301ADSCrossRefGoogle Scholar
  13. Brutscher B, Felli IC, Gil-Caballero S, Hošek T, Kümmerle R, Piai A, Pierattelli R, Sólyom Z (2015) NMR methods for the study of instrinsically disordered proteins structure, dynamics, and interactions: general overview and practical guidelines. Adv Exp Med Biol 870:122Google Scholar
  14. Dyson HJ, Wright PE (2001) Nuclear magnetic resonance methods for the elucidation of structure and dynamics in disordered states. Methods Enzymol 339:258–271CrossRefGoogle Scholar
  15. Emsley L, Bodenhausen G (1992) Optimization of shaped selective pulses for NMR using a quaternion description of their overall propagators. J Magn Reson 97:135–148ADSGoogle Scholar
  16. Felli IC, Pierattelli R (2015a) Spin-state-selctive methods in solution- and solid-state biomolecular 13C NMR. Prog NMR Spectrosc 84:1–13CrossRefGoogle Scholar
  17. Felli IC, Pierattelli R (eds) (2015b) Intrisically disordered proteins studied by NMR spectroscopy. Springer, SwitzerlandGoogle Scholar
  18. Felli IC, Pierattelli R, Glaser SJ, Luy B (2009) Relaxation-optimised Hartmann-Hahn transfer for carbonyl-carbonyl correlation spectroscopy using a specifically tailored MOCCA-XY16 mixing sequence for protonless 13C direct detection experiments. J Biomol NMR 43:187–196CrossRefGoogle Scholar
  19. Fiorito F, Hiller S, Wider G, Wüthrich K (2006) Automated resonance assignment of proteins: 6D APSY-NMR. J Biomol NMR 35:27–37CrossRefGoogle Scholar
  20. 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 52:11808–11812CrossRefGoogle Scholar
  21. Haba NY, Gross R, Nováček J, Shaked H, Židek L, Barda-Saad M, Chill JH (2013) NMR determines transient structure and dynamics in the disordered C-terminal domain of WASp interacting protein. Biophys J 105:481–493CrossRefGoogle Scholar
  22. Habchi J, Tompa P, Longhi S, Uversky VN (2014) Introducing protein intrinsic disorder. Chem Rev 114:6561–6588CrossRefGoogle Scholar
  23. Heller GT, Aprile FA, Vendruscolo M (2017) Methods of probing the interactions between small molecules and disordered proteins. Cell Mol Life Sci 74:3225–3243CrossRefGoogle Scholar
  24. Hiller S, Wider G (2012) Automated projection spectroscopy and its applications. Top Curr Chem 316:21–47CrossRefGoogle Scholar
  25. Hiller S, Fiorito F, Wüthrich K, Wider G (2005) Automated projection spectroscopy (APSY). Proc Natl Acad Sci USA 102:10876–10881ADSCrossRefGoogle Scholar
  26. Hiller S, Wider G, Wüthrich K (2008) APSY-NMR with proteins: practical aspects and backbone assignment. J Biomol NMR 42:179–195CrossRefGoogle Scholar
  27. Hoch JC, Stern AS (2001) Nuclear magnetic resonance of biological macromolecules. Academic Press, Cambridge, pp 159–178Google Scholar
  28. 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 42:173–177CrossRefGoogle Scholar
  29. Joshi P, Chia S, Habchi J, Knowles TPJ, Dobson CM, Vendruscolo M (2016) A fragment-based method of creating small-molecule libraries to target the aggregation of intrinsically disordered proteins. ACS Comb Sci 18:144–153CrossRefGoogle Scholar
  30. Kadkhodaie M, Rivas O, Tan M, Mohebbi A, Shaka AJ (1991) Broadband homonuclear cross polarization using flip-flop spectroscopy. J Magn Reson 91:437–443ADSGoogle Scholar
  31. Kay LE, Ikura M, Tschudin R, Bax A (1990) Three-dimensional triple-resonance NMR spectroscopy of isotopically enriched proteins. J Magn Reson 89:496–514ADSGoogle Scholar
  32. Kazimierczuk K, Zawadzka A, Koźmiński W, Zhukov I (2006) Random sampling of evolution time space and Fourier transform processing. J Biomol NMR 36:157–168CrossRefGoogle Scholar
  33. Kim S, Szyperski T (2003) GFT NMR, a new approach to rapidly obtain precise high-dimensional NMR spectral information. J Am Chem Soc 125:1385–1393CrossRefGoogle Scholar
  34. Kovacs H, Moskau D, Spraul M (2005) Cryogenically cooled probes: a leap in NMR technology. Prog NMR Spectrosc 46:131–155CrossRefGoogle Scholar
  35. Kupce E, Freeman R (2004) Projection-reconstruction technique for speeding up multidimensional NMR spectroscopy. J Am Chem Soc 126:6429–6440CrossRefGoogle Scholar
  36. 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 49:99–109CrossRefGoogle Scholar
  37. Markley JL, Bax A, Arata Y, Hilbers CW, Kaptein R, Sykes BD, Wright PE, Wüthrich K (1998) Recommendations for the presentation of NMR structures of proteins and nucleic acids. IUPAC-IUPMB-IUPAB inter-union task group on the standardization of data bases of protein and nucleic acid structures determined by NMR spectroscopy. Eur J Biochem 256:1–15CrossRefGoogle Scholar
  38. Motackova 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 48:169–177CrossRefGoogle Scholar
  39. Narayanan RL, Duerr 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 132:11906–11907CrossRefGoogle Scholar
  40. 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 50:1–11CrossRefGoogle Scholar
  41. Nováček J, Haba NY, Chill JH, Židek L, Sklenář V (2012) 4D non-uniformly sampled HCBCACON and 1JNCα-selective HCBCANCO experiments for the sequential assignment and chemical shift analysis of intrinsically disordered proteins. J Biomol NMR 53:139–148CrossRefGoogle Scholar
  42. 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 200:354–358ADSCrossRefGoogle Scholar
  43. Orekhov VY, Ibraghimov I, Billeter M (2003) Optimizing resolution in multidimensional NMR by three-way decomposition. J Biomol NMR 27:165–173CrossRefGoogle Scholar
  44. Piai A, Hošek T, Gonnelli L, Zawadzka-Kazimierczuk A, Koźmiński W, Brutscher B, Bermel W, Pierattelli R, Felli IC (2014) “CON–CON” assignment strategy for highly flexible intrinsically disordered proteins. J Biomol NMR 60:209–218CrossRefGoogle Scholar
  45. 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 34:93–158CrossRefGoogle Scholar
  46. Shaka AJ, Barker PB, Freeman R (1985) Computer-optimized decoupling scheme for wideband applications and low-level operation. J Magn Reson 64:547–552ADSGoogle Scholar
  47. Tóth G, Gardai SJ, Zago W, Bertoncini CW, Cremades N, Roy SL, Tambe MA, Rochet JC, Galvagnion C, Skibinski G, Finkbeiner S, Bova M, Regnstrom K, Chiou SS, Johnston J, Callaway K, Anderson JP, Jobling MF, Buell AK, Yednock TA, Knowles TP, Vendruscolo M, Christodoulou J, Dobson CM, Schenk D, McConlogue L (2014) Targeting the intrinsically disordered structural ensemble of α-synuclein by small molecules as a potential therapeutic strategy for Parkinson’s disease. PLoS ONE 9:e87133ADSCrossRefGoogle Scholar
  48. Uversky V, Oldfield CJ, Dunker AK (2008) Intrinsically disordered proteins in human diseases: introducing the D2 concept. Annu Rev Biophys 37:215–246CrossRefGoogle Scholar
  49. Uversky VN, Davé V, Iakoucheva LM, Malaney P, Metallo SJ, Pathak RR, Joerger AC (2014) Pathological unfoldomics of uncontrolled chaos: intrinsically disordered proteins and human diseases. Chem Rev 114:6844–6879CrossRefGoogle Scholar
  50. van der Lee R, Buljan M, Lang B, Weatheritt RJ, Daughdrill GW, Dunker AK, Fuxreiter M, Gough J, Gsponer J, Jones DT, Kim PM, Kriwacki RW, Oldfield CJ, Pappu RV, Tompa P, Uversky VN, Wright PE, Babu MM (2014) Classification of intrinsically disordered regions and proteins. Chem Rev 114:6589–6631CrossRefGoogle Scholar
  51. Wright PE, Dyson HJ (2015) Intrinsically disordered proteins in cellular signalling and regulation. Nat Rev Mol Cell Biol 16:18–29CrossRefGoogle Scholar
  52. Yao X, Stefan B, Zweckstetter M (2014) A six-dimensional alpha proton detection-based APSY experiment for backbone assignment of intrinsically disordered proteins. J Biomol NMR 60:231–240CrossRefGoogle Scholar
  53. Zawadzka-Kazimierczuk A, Kazimierczuk K, Koźmiński W (2010) A set of 4D NMR experiments of enhanced resolution for easy resonance assignment in proteins. J Magn Reson 202:109–116ADSCrossRefGoogle Scholar
  54. Zawadzka-Kazimierczuk A, Koźmiński W, Sanderová H, Krásný L (2012) High dimensional and high resolution pulse seqeunces for backbone resonance assignment of intrinsically disordered proteins. J Biomol NMR 52:329–337CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V., part of Springer Nature 2018

Authors and Affiliations

  • Maria Grazia Murrali
    • 1
  • Marco Schiavina
    • 1
  • Valerio Sainati
    • 1
  • Wolfgang Bermel
    • 2
  • Roberta Pierattelli
    • 1
    • 3
  • Isabella C. Felli
    • 1
    • 3
  1. 1.CERM, University of FlorenceSesto Fiorentino, FlorenceItaly
  2. 2.Bruker BioSpin GmbHRheinstettenGermany
  3. 3.Department of Chemistry “Ugo Schiff”University of FlorenceSesto Fiorentino, FlorenceItaly

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