Analyzing Temperature-Induced Transitions in Disordered Proteins by NMR Spectroscopy and Secondary Chemical Shift Analyses

  • Magnus Kjaergaard
  • Flemming M. Poulsen
  • Birthe B. KragelundEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 896)


Intrinsically disordered proteins are abundant in nature and perform many important physiological functions. Multidimensional NMR spectroscopy has been crucial for the understanding of the conformational properties of disordered proteins and is increasingly used to probe their conformational ensembles. Compared to folded proteins, disordered proteins are more malleable and more easily perturbed by environmental factors. Accordingly, the experimental conditions and especially the temperature modify the structural and functional properties of disordered proteins. This chapter discusses practical aspects of NMR studies of temperature-induced structural changes in disordered proteins using chemical shifts.

Key words

Intrinsically disordered protein Transient secondary structure Temperature dependence Random coil chemical shifts 



We thank our colleagues at SBiN-lab for sharing their practical experiences obtained from working with IDPs.


  1. 1.
    Uversky VN (2009) Intrinsically disordered proteins and their environment: effects of strong denaturants, temperature, pH, counter ions, membranes, binding partners, osmolytes, and macromolecular crowding. Protein J 28(7–8):305–325PubMedCrossRefGoogle Scholar
  2. 2.
    Jarvet J, Damberg P, Danielsson J, Johansson I, Eriksson LE, Graslund A (2003) A left-handed 3(1) helical conformation in the Alzheimer Abeta(12-28) peptide. FEBS Lett 555(2):371–374PubMedCrossRefGoogle Scholar
  3. 3.
    Uversky VN, Li J, Fink AL (2001) Evidence for a partially folded intermediate in alpha-synuclein fibril formation. J Biol Chem 276(14):10737–10744PubMedCrossRefGoogle Scholar
  4. 4.
    Dawson R, Muller L, Dehner A, Klein C, Kessler H, Buchner J (2003) The N-terminal domain of p53 is natively unfolded. J Mol Biol 332(5):1131–1141PubMedCrossRefGoogle Scholar
  5. 5.
    Gast K, Zirwer D, Damaschun G (2003) Are there temperature-dependent structural transitions in the “intrinsically unstructured” protein prothymosin alpha? Eur Biophys J 31(8):586–594PubMedGoogle Scholar
  6. 6.
    Jeganathan S, von Bergen M, Mandelkow EM, Mandelkow E (2008) The natively unfolded character of tau and its aggregation to Alzheimer-like paired helical filaments. Biochemistry 47(40):10526–10539PubMedCrossRefGoogle Scholar
  7. 7.
    Sanchez-Puig N, Veprintsev DB, Fersht AR (2005) Human full-length Securin is a natively unfolded protein. Protein Sci 14(6):1410–1418PubMedCrossRefGoogle Scholar
  8. 8.
    Malm J, Jonsson M, Frohm B, Linse S (2007) Structural properties of semenogelin I. FEBS J 274(17):4503–4510PubMedCrossRefGoogle Scholar
  9. 9.
    Yang WY, Larios E, Gruebele M (2003) On the extended beta-conformation propensity of polypeptides at high temperature. J Am Chem Soc 125(52):16220–16227PubMedCrossRefGoogle Scholar
  10. 10.
    Kjaergaard M, Norholm AB, Hendus-Altenburger R, Pedersen SF, Poulsen FM, Kragelund BB (2010) Temperature-dependent structural changes in intrinsically disordered proteins: formation of alpha-helices or loss of polyproline II? Protein Sci 19(8):1555–1564PubMedCrossRefGoogle Scholar
  11. 11.
    Kim HY, Heise H, Fernandez CO, Baldus M, Zweckstetter M (2007) Correlation of amyloid fibril beta-structure with the unfolded state of alpha-synuclein. Chembiochem 8(14):1671–1674PubMedCrossRefGoogle Scholar
  12. 12.
    Wu KP, Kim S, Fela DA, Baum J (2008) Characterization of conformational and dynamic properties of natively unfolded human and mouse alpha-synuclein ensembles by NMR: implication for aggregation. J Mol Biol 378(5):1104–1115PubMedCrossRefGoogle Scholar
  13. 13.
    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(21):7222–7223PubMedCrossRefGoogle Scholar
  14. 14.
    Modig K, Jurgensen VW, Lindorff-Larsen K, Fieber W, Bohr HG, Poulsen FM (2007) Detection of initiation sites in protein folding of the four helix bundle ACBP by chemical shift analysis. FEBS Lett 581(25):4965–4971PubMedCrossRefGoogle Scholar
  15. 15.
    Pace CN (1986) Determination and analysis of urea and guanidine hydrochloride denaturation curves. In: Hirs CHW, Timasheff SN (eds). Methods in enzymology, vol 131. Academic, Waltham, MA, pp 266–280Google Scholar
  16. 16.
    Findeisen M, Brand T, Berger S (2007) A 1H-NMR thermometer suitable for cryoprobes. Magn Reson Chem 45(2):175–178PubMedCrossRefGoogle Scholar
  17. 17.
    Eliezer D (2007) Characterizing residual structure in disordered protein states using nuclear magnetic resonance. Methods Mol Biol 350:49–67PubMedGoogle Scholar
  18. 18.
    Wishart DS, Bigam CG, Yao J, Abildgaard F, Dyson HJ, Oldfield E, Markley JL, Sykes BD (1995) 1H, 13C and 15N chemical shift referencing in biomolecular NMR. J Biomol NMR 6(2):135–140PubMedCrossRefGoogle Scholar
  19. 19.
    Grzesiek S, Bax A (1992) Improved 3D triple-resonance NMR techniques applied to a 31-kDa protein. J Magn Reson 96(2):432–440Google Scholar
  20. 20.
    Ikura M, Kay LE, Bax A (1990) A novel approach for sequential assignment of 1H, 13C, and 15N spectra of proteins: heteronuclear triple-resonance three-dimensional NMR spectroscopy. Application to calmodulin. Biochemistry 29(19):4659–4667PubMedCrossRefGoogle Scholar
  21. 21.
    Wittekind M, Mueller L (1993) Hncacb, a high-sensitivity 3D NMR experiment to correlate amide-proton and nitrogen resonances with the alpha-carbon and beta-carbon resonances in proteins. J Magn Reson Ser B 101(2):201–205CrossRefGoogle Scholar
  22. 22.
    Kay LE, Xu GY, Yamazaki T (1994) Enhanced-sensitivity triple-resonance spectroscopy with minimal H2O saturation. J Magn Reson Ser A 109(1):129–133CrossRefGoogle Scholar
  23. 23.
    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(16):6291–6293CrossRefGoogle Scholar
  24. 24.
    Panchal SC, Bhavesh NS, Hosur RV (2001) Improved 3D triple resonance experiments, HNN and HN(C)N, for HN and 15N sequential correlations in (13C, 15N) labeled proteins: application to unfolded proteins. J Biomol NMR 20(2):135–147PubMedCrossRefGoogle Scholar
  25. 25.
    Kjaergaard M, Brander S, Poulsen FM (2011) Random coil chemical shift for intrinsically disordered proteins: effects of temperature and pH. J Biomol NMR 49(2):139–49.Google Scholar
  26. 26.
    Tamiola K, Bi A, Mulder FAA (2010) Sequence-specific random coil chemical shifts of intrinsically disordered proteins. J Am Chem Soc 132(51):18000–18003PubMedCrossRefGoogle Scholar
  27. 27.
    Lam SL, Hsu VL (2003) NMR identification of left-handed polyproline type II helices. Biopolymers 69(2):270–281PubMedCrossRefGoogle Scholar
  28. 28.
    Iwadate M, Asakura T, Williamson MP (1999) C alpha and C beta carbon-13 chemical shifts in proteins from an empirical database. J Biomol NMR 13(3):199–211PubMedCrossRefGoogle Scholar
  29. 29.
    Zhang H, Neal S, Wishart DS (2003) RefDB: a database of uniformly referenced protein chemical shifts. J Biomol NMR 25(3):173–195PubMedCrossRefGoogle Scholar
  30. 30.
    Dawson RMC, Elliot DC, Elliot WH, Jones KM (1986) Data for biochemical research, 3rd edn. Oxford University Press, Oxford, UKGoogle Scholar
  31. 31.
    Teilum K, Kragelund BB, Poulsen FM (2005) Application of hydrogen exchange kinetics to studies of protein folding. Protein folding handbook. Wiley-VCH Verlag GmbH, Hoboken, New JerseyGoogle Scholar
  32. 32.
    Mantylahti S, Aitio O, Hellman M, Permi P (2010) HA-detected experiments for the backbone assignment of intrinsically disordered proteins. J Biomol NMR 47(3):171–181PubMedCrossRefGoogle Scholar
  33. 33.
    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(12):3918–3919PubMedCrossRefGoogle Scholar
  34. 34.
    Demarest SJ, Martinez-Yamout M, Chung J, Chen H, Xu W, Dyson HJ, Evans RM, Wright PE (2002) Mutual synergistic folding in recruitment of CBP/p300 by p160 nuclear receptor coactivators. Nature 415(6871):549–553PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2012

Authors and Affiliations

  • Magnus Kjaergaard
    • 1
  • Flemming M. Poulsen
    • 1
  • Birthe B. Kragelund
    • 1
    Email author
  1. 1.Structural Biology and NMR Laboratory, Department of BiologyUniversity of CopenhagenCopenhagenDenmark

Personalised recommendations