Skip to main content
SpringerLink
Log in

A six-dimensional alpha proton detection-based APSY experiment for backbone assignment of intrinsically disordered proteins

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

Abstract

Sequence specific resonance assignment is the prerequisite for the NMR-based analysis of the conformational ensembles and their underlying dynamics of intrinsically disordered proteins. However, rapid solvent exchange in intrinsically disordered proteins often complicates assignment strategies based on HN-detection. Here we present a six-dimensional alpha proton detection-based automated projection spectroscopy (APSY) experiment for backbone assignment of intrinsically disordered proteins. The 6D HCACONCAH APSY correlates the six different chemical shifts, Hα(i − 1), Cα(i − 1), C′(i − 1), N(i), Cα(i) and Hα(i). Application to two intrinsically disordered proteins, 140-residue α-synuclein and a 352-residue isoform of Tau, demonstrates that the chemical shift information provided by the 6D HCACONCAH APSY allows efficient backbone resonance assignment of intrinsically disordered proteins.

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

Similar content being viewed by others

References

  • Atreya HS, Szyperski T (2004) G-matrix Fourier transform NMR spectroscopy for complete protein resonance assignment. Proc Natl Acad Sci USA 101:9642–9647. doi:10.1073/pnas.0403529101

    Article  ADS  Google Scholar 

  • Barna JCJ, Laue ED, Mayger MR, Skilling J, Worrall SJP (1987) Exponential sampling, an alternative method for sampling in two-dimensional NMR experiments. J Magn Reson 73:69–77. doi:10.1016/0022-2364(87)90225-3

    ADS  Google Scholar 

  • Bermel W, Bertini I, Felli IC, Kummerle 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 178:56–64. doi:10.1016/j.jmr.2005.08.011

    Article  ADS  Google Scholar 

  • Bermel W, Bertini I, Felli IC, Lee YM, Luchinat C, Pierattelli R (2006b) Protonless NMR experiments for sequence-specific assignment of backbone nuclei in unfolded proteins. J Am Chem Soc 128:3918–3919. doi:10.1021/Ja0582206

    Article  Google Scholar 

  • Bermel W, Bertini I, Chill J, Felli IC, Haba N, Kumar MVV, Pierattelli R (2012a) Exclusively heteronuclear C-13-Detected amino-acid-selective NMR experiments for the study of intrinsically disordered proteins (IDPs). Chembiochem 13:2425–2432. doi:10.1002/cbic.201200447

    Article  Google Scholar 

  • Bermel W et al (2012b) Speeding up sequence specific assignment of IDPs. J Biomol NMR 53:293–301. doi:10.1007/s10858-012-9639-0

    Article  Google Scholar 

  • Bermel W, Felli IC, Gonnelli L, Kozminski W, Piai A, Pierattelli R, Zawadzka-Kazimierczuk A (2013) High-dimensionality 13C direct-detected NMR experiments for the automatic assignment of intrinsically disordered proteins. J Biomol NMR 57:353–361. doi:10.1007/s10858-013-9793-z

    Article  Google Scholar 

  • Bertini I, Duma L, Felli IC, Fey M, Luchinat C, Pierattelli R, Vasos PR (2004) A heteronuclear direct-detection NMR spectroscopy experiment for protein-backbone assignment. Angew Chem Int Edit 43:2257–2259. doi:10.1002/anie.200453661

    Article  Google Scholar 

  • Bottomley MJ, Macias MJ, Liu Z, Sattler M (1999) A novel NMR experiment for the sequential assignment of proline residues and proline stretches in 13C/15 N-labeled proteins. J Biomol NMR 13:381–385

    Article  Google Scholar 

  • Braak H, Braak E (1991) Neuropathological stageing of Alzheimer-related changes. Acta Neuropathol 82:239–259

    Article  Google Scholar 

  • Chylla RA, Markley JL (1995) Theory and application of the maximum likelihood principle to NMR parameter estimation of multidimensional NMR data. J Biomol NMR 5:245–258

    Article  Google Scholar 

  • Coggins BE, Zhou P (2006) Polar Fourier transforms of radially sampled NMR data. J Magn Reson 182:84–95. doi:10.1016/j.jmr.2006.06.016

    Article  ADS  Google Scholar 

  • Coggins BE, Venters RA, Zhou P (2004) Generalized reconstruction of n-D NMR spectra from multiple projections: application to the 5-D HACACONH spectrum of protein G B1 domain. J Am Chem Soc 126:1000–1001. doi:10.1021/ja039430q

    Article  Google Scholar 

  • Croke RL, Sallum CO, Watson E, Watt ED, Alexandrescu AT (2008) Hydrogen exchange of monomeric alpha-synuclein shows unfolded structure persists at physiological temperature and is independent of molecular crowding in Escherichia coli. Protein Sci 17:1434–1445. doi:10.1110/ps.033803.107

    Article  Google Scholar 

  • Csizmok V, Felli IC, Tompa P, Banci L, Bertini I (2008) Structural and dynamic characterization of intrinsically disordered human securin by NMR spectroscopy. J Am Chem Soc 130:16873–16879. doi:10.1021/ja805510b

    Article  Google Scholar 

  • Felli IC, Brutscher B (2009) Recent advances in solution NMR: fast methods and heteronuclear direct detection. Chemphyschem 10:1356–1368. doi:10.1002/cphc.200900133

    Article  Google Scholar 

  • Fiorito F, Hiller S, Wider G, Wuthrich K (2006) Automated resonance assignment of proteins: 6D APSY-NMR. J Biomol NMR 35:27–37. doi:10.1007/s10858-006-0030-x

    Article  Google Scholar 

  • Güntert P, Dötsch V, Wider G, Wüthrich K (1992) Processing of multidimensional NMR data with the new software PROSA. J Biomol NMR 2:619–629. doi:10.1007/Bf02192850

    Article  Google Scholar 

  • Hiller S, Fiorito F, Wuthrich K, Wider G (2005) Automated projection spectroscopy (APSY). Proc Natl Acad Sci USA 102:10876–10881. doi:10.1073/pnas.0504818102

    Article  ADS  Google Scholar 

  • Hiller S, Wider G, Wuthrich K (2008) APSY-NMR with proteins: practical aspects and backbone assignment. J Biomol NMR 42:179–195. doi:10.1007/s10858-008-9266-y

    Article  Google Scholar 

  • Holland DJ, Bostock MJ, Gladden LF, Nietlispach D (2011) Fast multidimensional NMR spectroscopy using compressed sensing. Angew Chem Int Edit 50:6548–6551. doi:10.1002/Anie.201100440

    Article  Google Scholar 

  • Hu K, Vogeli B, Clore GM (2007) Spin-state selective carbon-detected HNCO with TROSY optimization in all dimensions and double echo-antiecho sensitivity enhancement in both indirect dimensions. J Am Chem Soc 129:5484–5491. doi:10.1021/ja067981l

    Article  Google Scholar 

  • Iakoucheva LM, Brown CJ, Lawson JD, Obradovic Z, Dunker AK (2002) Intrinsic disorder in cell-signaling and cancer-associated proteins. J Mol Biol 323:573–584

    Article  Google Scholar 

  • Jensen MR, Ruigrok RW, Blackledge M (2013) Describing intrinsically disordered proteins at atomic resolution by NMR. Curr Opin Struct Biol 23:426–435. doi:10.1016/j.sbi.2013.02.007

    Article  Google Scholar 

  • Jung YS, Zweckstetter M (2004) Mars—robust automatic backbone assignment of proteins. J Biomol NMR 30:11–23. doi:10.1023/B:JNMR.0000042954.99056.ad

    Article  Google Scholar 

  • Kanelis V, Donaldson L, Muhandiram DR, Rotin D, Forman-Kay JD, Kay LE (2000) Sequential assignment of proline-rich regions in proteins: application to modular binding domain complexes. J Biomol NMR 16:253–259. doi:10.1023/A:1008355012528

    Article  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. doi:10.1021/Ja00052a088

    Article  Google Scholar 

  • Kazimierczuk K, Orekhov VY (2011) Accelerated NMR spectroscopy by using compressed sensing. Angew Chem Int Ed Engl 50:5556–5559. doi:10.1002/anie.201100370

    Article  Google Scholar 

  • Kazimierczuk K, Kozminski W, Zhukov I (2006) Two-dimensional Fourier transform of arbitrarily sampled NMR data sets. J Magn Reson 179:323–328. doi:10.1016/j.jmr.2006.02.001

    Article  ADS  Google Scholar 

  • Kazimierczuk K, Stanek J, Zawadzka-Kazimierczuk A, Kozminski W (2013) High-dimensional NMR spectra for structural studies of biomolecules. Chemphyschem 14:3015–3025. doi:10.1002/cphc.201300277

    Article  Google Scholar 

  • Kim S, Szyperski T (2003) GFT NMR, a new approach to rapidly obtain precise high-dimensional NMR spectral information. J Am Chem Soc 125:1385–1393. doi:10.1021/Ja028197d

    Article  Google Scholar 

  • Korzhneva DM, Ibraghimov IV, Billeter M, Orekhov VY (2001) MUNIN: application of three-way decomposition to the analysis of heteronuclear NMR relaxation data. J Biomol NMR 21:263–268

    Article  Google Scholar 

  • Kupce E, Freeman R (2003a) Projection-reconstruction of three-dimensional NMR spectra. J Am Chem Soc 125:13958–13959. doi:10.1021/Ja038297z

    Article  Google Scholar 

  • Kupce E, Freeman R (2003b) Reconstruction of the three-dimensional NMR spectrum of a protein from a set of plane projections. J Biomol NMR 27:383–387

    Article  Google Scholar 

  • Kupce E, Freeman R (2004) Projection-reconstruction technique for speeding up multidimensional NMR spectroscopy. J Am Chem Soc 126:6429–6440. doi:10.1021/ja049432q

    Article  Google Scholar 

  • Mantylahti 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. doi:10.1007/s10858-010-9421-0

    Article  Google Scholar 

  • Mantylahti 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. doi:10.1007/s10858-011-9470-z

    Article  Google Scholar 

  • Marsh JA, Forman-Kay JD (2010) Sequence determinants of compaction in intrinsically disordered proteins. Biophys J 98:2383–2390. doi:10.1016/j.bpj.2010.02.006

    Article  Google Scholar 

  • Mccoy MA, Mueller L (1992) Nonresonant effects of frequency-selective pulses. J Magn Reson 99:18–36. doi:10.1016/0022-2364(92)90152-W

    ADS  Google Scholar 

  • Mittag T, Forman-Kay JD (2007) Atomic-level characterization of disordered protein ensembles. Curr Opin Struct Biol 17:3–14. doi:10.1016/j.sbi.2007.01.009

    Article  Google Scholar 

  • Motackova V et al (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. doi:10.1007/s10858-010-9447-3

    Article  Google Scholar 

  • Narayanan RL, Durr UH, Bibow S, Biernat J, Mandelkow E, Zweckstetter M (2010) Automatic assignment of the intrinsically disordered protein Tau with 441-residues. J Am Chem Soc 132:11906–11907. doi:10.1021/ja105657f

    Article  Google Scholar 

  • Novacek J et al (2011) 5D 13C-detected experiments for backbone assignment of unstructured proteins with a very low signal dispersion. J Biomol NMR 50:1–11. doi:10.1007/s10858-011-9496-2

    Article  Google Scholar 

  • Novacek J, Janda L, Dopitova R, Zidek L, Sklenar 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. doi:10.1007/s10858-013-9761-7

    Article  Google Scholar 

  • Orekhov VY, Ibraghimov IV, Billeter M (2001) MUNIN: a new approach to multi-dimensional NMR spectra interpretation. J Biomol NMR 20:49–60

    Article  Google Scholar 

  • Pervushin K, Eletsky A (2003) A new strategy for backbone resonance assignment in large proteins using a MQ-HACACO experiment. J Biomol NMR 25:147–152

    Article  Google Scholar 

  • Rezaei-Ghaleh N, Blackledge M, Zweckstetter M (2012) Intrinsically disordered proteins: from sequence and conformational properties toward drug discovery. Chembiochem 13:930–950. doi:10.1002/cbic.201200093

    Article  Google Scholar 

  • Shen Y, Atreya HS, Liu GH, Szyperski T (2005) G-matrix Fourier transform NOESY-based protocol for high-quality protein structure determination. J Am Chem Soc 127:9085–9099. doi:10.1021/Ja0501870

    Article  Google Scholar 

  • Shimba N et al (2004) Optimization of 13C direct detection NMR methods. J Biomol NMR 30:175–179. doi:10.1023/B:JNMR.0000048855.35771.11

    Article  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. doi:10.1021/ja100453e

    Article  Google Scholar 

  • Szyperski T, Atreya HS (2006) Principles and applications of GFT projection NMR spectroscopy. Magn Reson Chem 44:51–60. doi:10.1002/mrc.1817

    Article  Google Scholar 

  • Takeuchi K, Frueh DP, Hyberts SG, Sun ZY, Wagner G (2010) High-resolution 3D CANCA NMR experiments for complete mainchain assignments using Cα direct detection. J Am Chem Soc 132:2945–2951. doi:10.1021/ja907717b

    Article  Google Scholar 

  • Tompa P (2002) Intrinsically unstructured proteins. Trends Biochem Sci 27:527–533

    Article  Google Scholar 

  • Uversky VN (2002) Natively unfolded proteins: a point where biology waits for physics. Protein Sci 11:739–756. doi:10.1110/ps.4210102

    Article  Google Scholar 

  • Uversky VN (2011) Flexible nets of malleable guardians: intrinsically disordered chaperones in neurodegenerative diseases. Chem Rev 111:1134–1166. doi:10.1021/cr100186d

    Article  Google Scholar 

  • Uversky VN, Oldfield CJ, Dunker AK (2008) Intrinsically disordered proteins in human diseases: introducing the D2 concept. Annu Rev Biophys 37:215–246. doi:10.1146/annurev.biophys.37.032807.125924

    Article  Google Scholar 

  • Wang AC, Grzesiek S, Tschudin R, Lodi PJ, Bax A (1995) Sequential backbone assignment of isotopically enriched proteins in D2O by deuterium-decoupled HA(CA)N and HA(CACO)N. J Biomol NMR 5:376–382

    Google Scholar 

  • Wilkins DK, Grimshaw SB, Receveur V, Dobson CM, Jones JA, Smith LJ (1999) Hydrodynamic radii of native and denatured proteins measured by pulse field gradient NMR techniques. Biochemistry 38:16424–16431. doi:10.1021/Bi991765q

    Article  Google Scholar 

  • Wright PE, Dyson HJ (1999) Intrinsically unstructured proteins: re-assessing the protein structure-function paradigm. J Mol Biol 293:321–331. doi:10.1006/jmbi.1999.3110

    Article  Google Scholar 

  • Zawadzka-Kazimierczuk A, Kozminski 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. doi:10.1007/s10858-012-9652-3

    Article  Google Scholar 

  • Zawadzka-Kazimierczuk A, Kozminski W, Sanderova H, Krasny L (2012b) High dimensional and high resolution pulse sequences for backbone resonance assignment of intrinsically disordered proteins. J Biomol NMR 52:329–337. doi:10.1007/s10858-012-9613-x

    Article  Google Scholar 

Download references

Acknowledgments

We thank Eckhard Mandelkow and Jacek Biernat for the htau23 sample. This work was in part supported by the DFG through ZW71/3-2 and ZW71/7-1.

Author information

Authors and Affiliations

  1. Department for NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, 37077, Göttingen, Germany

    Xuejun Yao, Stefan Becker & Markus Zweckstetter

  2. German Center for Neurodegenerative Diseases (DZNE), Göttingen, 37077, Göttingen, Germany

    Markus Zweckstetter

  3. Center for Nanoscale Microscopy and Molecular Physiology of the Brain, University Medical Center Göttingen, 37075, Göttingen, Germany

    Markus Zweckstetter

Authors
  1. Xuejun Yao
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Stefan Becker
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Markus Zweckstetter
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Markus Zweckstetter.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 83 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Yao, X., Becker, S. & Zweckstetter, M. A six-dimensional alpha proton detection-based APSY experiment for backbone assignment of intrinsically disordered proteins. J Biomol NMR 60, 231–240 (2014). https://doi.org/10.1007/s10858-014-9872-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10858-014-9872-9

Keywords

Search

Navigation