Skip to main content
SpringerLink
Log in

HA-detected experiments for the backbone assignment of intrinsically disordered proteins

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

Abstract

We propose a new alpha proton detection based approach for the sequential assignment of natively unfolded proteins. The proposed protocol superimposes on following features: HA-detection (1) enables assignment of natively unfolded proteins at any pH, i.e., it is not sensitive to rapid chemical exchange undergoing in natively unfolded proteins even at moderately high pH. (2) It allows straightforward assignment of proline-rich polypeptides without additional proline-customized experiments. (3) It offers more streamlined and less ambiguous assignment based on solely intraresidual 15N(i)-13C′(i)-Hα(i) (or 15N(i)-13Cα(i)-Hα(i)) and sequential 15N(i + 1)-13C′(i)-Hα(i) (or 15N(i + 1)-13Cα(i)-Hα(i)) correlation experiments together with efficient use of chemical shifts of 15N and 13C′ nuclei, which show smaller dependence on residue type. We have tested the proposed protocol on two proteins, small globular 56-residue GB1, and highly disordered, proline-rich 47-residue fifth repeat of EspFU. Using the proposed approach, we were able to assign 90% of 1Hα, 13Cα, 13C′, 15N chemical shifts in EspFU. We reckon that the HA-detection based strategy will be very useful in the assignment of natively unfolded proline-rich proteins or polypeptide chains.

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

Similar content being viewed by others

References

  • Alho N, Klaavuniemi T, Ylanne J, Permi P, Mattila S (2007) Backbone NMR assignment of the internal interaction site of ALP. Biomol NMR Assign 1:85–87

    Article  Google Scholar 

  • Bai Y, Milne JS, Mayne L, Englander SW (1993) Primary structure effects on peptide group hydrogen exchange. Proteins Struct Funct Genet 17:75–86

    Article  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

    Google Scholar 

  • Bendall MR (1995) Heteronuclear J coupling precession during spin-lock and adiabatic pulses. Use of adiabatic inversion pulses in high-resolution NMR. J Magn Reson A116:46–58

    ADS  Google Scholar 

  • Bermel W, Bertini I, Felli IC, Piccioli M, Pierattelli R (2006) 13C-detected protonless NMR spectroscopy of proteins in solution. Prog Nucl Magn Reson Spectr 48:25–45

    Article  Google Scholar 

  • Bermel W, Bertini I, Csizmok V, Felli IC, Pierattelli R, Tompa P (2009) H-start for exclusively heteronuclear NMR spectroscopy: the case of intrinsically disordered protein. J Magn Reson 198:275–281

    Article  ADS  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/15N-labeled proteins. J Biomol NMR 13:381–385

    Article  Google Scholar 

  • Brutscher B (2002) Intraresidue HNCA and COHNCA experiments for protein backbone resonance assignment. J Magn Reson 156:155–159

    Article  ADS  Google Scholar 

  • Cheng HC, Skehan BM, Campellone KG, Leong JM, Rosen MK (2008) Structural mechanism of WASP activation by the enterohaemorrhagic E. coli effector EspF(U). Nature 454:1009–1013

    Article  ADS  Google Scholar 

  • Coggins BE, Zhou P (2008) High resolution 4-D spectroscopy with sparse concentric shell sampling and FFT-CLEAN. J Biomol NMR 42:225–239

    Article  Google Scholar 

  • Delaglio F, Torchia DA, Bax A (1991) Measurement of 15N–13C J couplings in staphylococcal nuclease. J Biomol NMR 1:439–446

    Article  Google Scholar 

  • Dyson HJ, Wright PE (2001) Nuclear magnetic resonance methods for elucidation of structure and dynamics in disordered states. Methods Enzymol 339:258–270

    Article  Google Scholar 

  • Dyson HJ, Wright PE (2005) Intrinsically unstructured proteins and their functions. Nat Rev Mol Cell Biol 6:197–208

    Article  Google Scholar 

  • Freeman R, Kupče E (2004) Distant echoes of the accordion: reduced dimensionality, GFT-NMR, and projection-reconstruction of multidimensional spectra. Concepts Magn Reson 23A:63–75

    Article  Google Scholar 

  • Goddard TD, Kneller DG (2004) SPARKY 3. University of California, San Francisco

  • Grzesiek S, Bax A, Hu J-S, Kaufman J, Palmer I, Stahl SJ, Tjandra N, Wingfield PT (1997) Refined solution structure and backbone dynamics of HIV-1 Nef. Prot Sci 6:1248–1263

    Article  Google Scholar 

  • Hu K, Vögeli 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

    Article  Google Scholar 

  • Jaravine VA, Zhuravleva AV, Permi P, Ibraghimov I, Orekhov VY (2008) Hyperdimensional NMR spectroscopy with nonlinear sampling. J Am Chem Soc 130:3927–3936

    Article  Google Scholar 

  • Jiang L, Coggins BE, Zhou P (2005) Rapid assignment of protein side chain resonances using projection-reconstruction of (4, 3)D HC(CCO)NH and intra-HC(C)NH experiments. J Magn Reson 175:170–176

    Article  ADS  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

    Article  Google Scholar 

  • Kay LE, Ikura M, Tschudin R, Bax A (1990) Three-dimensional triple-resonance NMR spectroscopy of isotopically enriched proteins. J Magn Reson 89:496–514

    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 

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

    Article  Google Scholar 

  • Kupće E, Wagner G (1995) Wideband homonuclear decoupling in protein spectra. J Magn Reson 109A:329–333

    Google Scholar 

  • Mäntylahti S, Tossavainen H, Hellman M, Permi P (2009) An intraresidual i(HCA)CO(CA)NH experiment for the assignment of main-chain resonances in 15N, 13C labeled proteins. J Biomol NMR 45:301–310

    Article  Google Scholar 

  • Marion D (2006) Processing of ND NMR spectra sampled in polar coordinates: a simple Fourier transform instead of a reconstruction. J Biomol NMR 36:45–54

    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

    Google Scholar 

  • Morris GA, Freeman R (1979) Enhancement of nuclear magnetic resonance signals by polarization transfer. J Am Chem Soc 101:760–762

    Article  Google Scholar 

  • Nietlispach D (2004) A selective intra-HN(CA)CO experiment for the backbone assignment of deuterated proteins. J Biomol NMR 28:131–136

    Article  Google Scholar 

  • Nietlispach D, Ito Y, Laue ED (2002) A novel approach for the sequential backbone assignment of larger proteins: selective intra-HNCA and DQ-HNCA. J Am Chem Soc 124:11199–11207

    Article  Google Scholar 

  • Permi P (2002) Intraresidual HNCA: an experiment for correlating only intraresidual backbone resonances. J Biomol NMR 23:201–209

    Article  Google Scholar 

  • Permi P, Annila A (2004) Coherence transfer in proteins. Prog Nucl Magn Reson Spectr 44:97–137

    Article  Google Scholar 

  • Pervushin K, Riek R, Wider G, Wüthrich K (1997) Attenuated T2 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 U S A 94:12366–12731

    Article  ADS  Google Scholar 

  • Powers R, Gronenborn AM, Clore GM, Bax A (1991) Three-dimensional triple-resonance NMR of 13C/15N-enriched proteins using constant-time evolution. J Magn Reson 94:209–213

    Google Scholar 

  • Rovnyak D, Frueh DP, Sastry M, Sun Z-YJ, Stern AS, Hoch JC, Wagner G (2004) Accelerated acquisition of high resolution triple-resonance spectra using non-uniform sampling and maximum entropy reconstruction. J Magn Reson 170:15–21

    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 Spectr 34:93–158

    Article  Google Scholar 

  • Sayers EW, Torchia DA (2001) Use of the carbonyl chemical shift to relieve degeneracies in triple-resonance assignment experiments. J Magn Reson 153:246–253

    Article  ADS  Google Scholar 

  • Schleucher J, Schwendinger MG, Sattler M, Schmidt P, Glaser SJ, Sørensen OW, Griesinger C (1994) A general enhancement scheme in heteronuclear multidimensional NMR employing pulsed-field gradients. J Biomol NMR 4:301–306

    Article  Google Scholar 

  • Shaka AJ (1985) Composite pulses for ultra-broadband spin inversion. Chem Phys Lett 120:201–205

    Article  ADS  Google Scholar 

  • Shaka AJ, Keeler J, Frenkiel T, Freeman R (1983) An improved sequence for broad-band decoupling–Waltz-16. J Magn Reson 52:335–338

    Google Scholar 

  • Silver MS, Joseph RJ, Hoult DI (1984) Highly selective π/2 and π pulse generation. J Magn Reson 59:347–351

    Google Scholar 

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

    Article  Google Scholar 

  • Tossavainen H, Permi P (2004) Optimized pathway selection in intraresidual triple-resonance experiments. J Magn Reson 170:244–251

    Article  ADS  Google Scholar 

  • Varian Inc. NMR Systems (2006) Varian NMR Systems with VNMRJ Software

  • Wang AC, Bax A (1995) Reparametrization of the Karplus relation for 3 J(Hα–N) and 3 J(HN–C′) in peptides from uniformly 13C/15N-enriched human ubiquitin. J Am Chem Soc 117:1810–1813

    Article  Google Scholar 

  • Wang A, Grzesiek S, Tschudin R, Lodi PJ, Bax A (1995) Sequential backbone assignment of isotopically enriched proteins in D20 by deuterium-decoupled HA(CA)N and HA(CACO)N. J Biomol NMR 5:376–382

    Google Scholar 

  • Yamazaki T, Lee W, Arrowsmith CH, Muhandiram DR, Kay LE (1994) A suite of triple-resonance NMR experiments for the backbone assignment of 15N, 13C, 2H labeled proteins with high-sensitivity. J Am Chem Soc 116:11655–11666

    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 

Download references

Acknowledgments

This work was financially supported by the grants 122170 and 131144 (to P. P.) from the Academy of Finland. We thank Vytautas Raulinaitis for useful discussions, and Elina Ahovuo and Jessica Buchmüller for excellent technical assistance.

Author information

Authors and Affiliations

  1. Program in Structural Biology and Biophysics, Institute of Biotechnology/NMR Laboratory, University of Helsinki, P.O. Box 65, 00014, Helsinki, Finland

    Sampo Mäntylahti, Olli Aitio, Maarit Hellman & Perttu Permi

Authors
  1. Sampo Mäntylahti
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Olli Aitio
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Maarit Hellman
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Perttu Permi
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Perttu Permi.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 14 kb)

Supplementary material 2 (TIFF 22173 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Mäntylahti, S., Aitio, O., Hellman, M. et al. HA-detected experiments for the backbone assignment of intrinsically disordered proteins. J Biomol NMR 47, 171–181 (2010). https://doi.org/10.1007/s10858-010-9421-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10858-010-9421-0

Keywords

Search

Navigation