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5D 13C-detected experiments for backbone assignment of unstructured proteins with a very low signal dispersion

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Abstract

Two novel 5D NMR experiments (CACONCACO, NCOCANCO) for backbone assignment of disordered proteins are presented. The pulse sequences exploit relaxation properties of the unstructured proteins and combine the advantages of 13C-direct detection, non-uniform sampling, and longitudinal relaxation optimization to maximize the achievable resolution and minimize the experimental time. The pulse sequences were successfully tested on the sample of partially disordered delta subunit from RNA polymerase from Bacillus subtilis. The unstructured part of this 20 kDa protein consists of 81 amino acids with frequent sequential repeats. A collection of 0.0003% of the data needed for a conventional experiment with linear sampling was sufficient to perform an unambiguous assignment of the disordered part of the protein from a single 5D spectrum.

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References

  • Achberger EC, Hilton MD, Whiteley HR (1982) The effect of the delta subunit on the interaction of Bacillus subtilis RNA polymerase with bases in a SP82 early gene promoter. Nucl Acids Res 10:2893–2910

    Article  Google Scholar 

  • Atreya HS, Szyperski T (2004) G-matrix Fourier transform NMR spectroscopy for complete protein resonance assignment. PNAS 101(26):9642–9647

    Article  ADS  Google Scholar 

  • Atreya H, Eletsky A, Szyperski T (2005) Resonance assignment of proteins with high shift degeneracy based on 5D spectral information encoded in G(2)FT NMR experiments. J Am Chem Soc 127(13):4554–4555

    Article  Google Scholar 

  • Bermel W, Bertini I, Duma L, Felli IC, Emsley L, Pierattelli R, Vasos PR (2005) Complete assignment of heteronuclear protein resonances by protonless NMR spectroscopy. Angew Chem Int Ed 44(20):3089–3092

    Article  Google Scholar 

  • Bermel W, Bertini I, Felli IC, Piccioli M, Pierattelli R (2006) C-13-detected protonless NMR spectroscopy of proteins in solution. Prog Nucl Magn Reson Spectrosc 48(1):25–45

    Article  Google Scholar 

  • Bermel W, Bertini I, Felli I, Lee Y, Luchinat C, Pierattelli R (2006) Protonless NMR experiments for sequence-specific assignment of backbone nuclei in unfolded proteins. J Am Chem Soc 128(12):3918–3919

    Article  Google Scholar 

  • Bermel W, Bertini I, Felli IC, Piccioli M, Pierattelli R (2009) Speeding up C-13 direct detection biomolecular NMR spectroscopy. J Am Chem Soc 131(42):15339–15345

    Article  Google Scholar 

  • Bretthorst GL (2008) Nonuniform sampling: bandwidth and aliasing. Concepts Magn Reson 32A(6):417–435

    Article  Google Scholar 

  • Brutscher B (2002) Intraresidue HNCA and COHNCA experiments for protein backbone resonance assignment. J Magn Reson 156(1):155–159

    Article  ADS  Google Scholar 

  • Delaglio F, Grzesiek S, Vuister G, Zhu G, Pfeifer J, Bax A (1995) NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR 6(3):277–293

    Article  Google Scholar 

  • Dunker AK, Obradovic Z, Romero P, Garner EC, Brown CJ (2000) Intrinsic protein disorder in complete genomes. Genome Inform 11:161–171

    Google Scholar 

  • Eliezer D (2007) Characterizing residual structure in disordered protein states using nuclear magnetic resonance. Methods Mol Biol 350:49–67

    Google Scholar 

  • Fiorito F, Hiller S, Wider G, Wüthrich K (2006) Automated resonance assignment of proteins: 6D APSY-NMR. J Biomol NMR 35(1):27–37

    Article  Google Scholar 

  • Freeman R, Kupče E (2004) Distant echoes of the accordion: reduced dimensionality, GFT-NMR, and projection-reconstruction of multidimensional spectra. Concepts Mang Reson 23(2):63–75

    Article  Google Scholar 

  • Frueh DP, Sun ZYJ, Vosburg DA, Walsh CT, Hoch JC, Wagner G (2006) Non-uniformly sampled double-TROSY hNcaNH experiments for NMR sequential assignments of large proteins. J Am Chem Soc 128(17):5757–5763

    Article  Google Scholar 

  • Hiller S, Fiorito F, Wuthrich K, Wider G (2005) Automated projection spectroscopy (APSY). PNAS 102(31):10876–10881

    Article  ADS  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(35):10823–10828

    Article  Google Scholar 

  • Kazimierczuk K, Zawadzka A, Koźmiński W, Zhukov I (2007) Lineshapes and artifacts in Multidimensional Fourier Transform of arbitrary sampled NMR data sets. J Magn Reson 188(2):344–356

    Article  ADS  Google Scholar 

  • Kazimierczuk K, Zawadzka A, Koźmiński W (2008) Optimization of random time domain sampling in multidimensional NMR. J Magn Reson 192(1):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 205(2):286–292

    Article  ADS  Google Scholar 

  • Kazimierczuk K, Zawadzka-Kazimierczuk A, Koźmiński W (2010) Non-uniform frequency domain for optimal exploitation of non-uniform sampling. J Magn Reson 197(2):219–228

    Article  ADS  Google Scholar 

  • Knoblich K, Whittaker S, Ludwig C, Michiels P, Jiang T, Schaffhausen B, Guenther U (2009) Backbone assignment of the N-terminal polyomavirus large T antigen. Biomol NMR Assign 3(1):119–123

    Article  Google Scholar 

  • Malmodin D, Billeter M (2005) Multiway decomposition of NMR spectra with coupled evolution periods. J Am Chem Soc 127(39):13486–13487

    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(1):45–54

    Article  Google Scholar 

  • Mobli M, Hoch JC (2008) Maximum entropy spectral reconstruction of nonuniformly sampled data. Concepts Magn Reson 32A(6):436–448

    Article  Google Scholar 

  • Motáčková V, Kubíčková M, Kožíšek M, Grantz-Šašková K, Švec M, Žídek L, Sklenář V (2009) Backbone H-1, C-13, and N-15 NMR assignment for the inactive form of the retroviral protease of the murine intracisternal A-type particle, inMIA-14 PR. Biomol NMR Assign 3(2):261–264

    Article  Google Scholar 

  • Motáčková V, Šanderová H, Žídek L, Nováček J, Padrta P, Švenková A, Korelusová J, Jonák J, Krásný L, Sklenář V (2010) Solution structure of the N-terminal domain of Bacillus subtilis delta subunit of RNA polymerase and its classification based on structural homologs. Proteins Struct Funct Bioinf 78(7):1807–1810

    Google Scholar 

  • Motáčková V, Nováček J, Zawadzka-Kazimierczuk A, Kazimierczuk K, Žídek 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(3):169–177

    Article  Google Scholar 

  • Mukrasch M, Bibow S, Korukottu J, Jeganathan S, Biernat J, Griesinger C, Mandelkow E, Zweckstetter M (2009) Structural polymorphism of 441-residue Tau at single sesidue resolution. PLoS Biol 7(2):399–414

    Article  Google Scholar 

  • Narayanan RL, Durr UHN, Bibow S, Biernat J, Mandelkow E3, Zweckstetter M (2010) Automatic assignment of the intrinsically disordered protein tau with 441-residues. J Am Chem Soc 132(34):11906–11907

    Article  Google Scholar 

  • Orekhov VY, Ibraghimov IV, Billeter M (2001) MUNIN: a new approach to multi-dimensional NMR spectra interpretation. J Biomol NMR 20(1):49–60

    Article  Google Scholar 

  • Panchal SC, Bhavesh NS, Hosur RV (2001) Improved 3D triple resonance experiments, HNN and HN(C)N, for H-N and N-15 sequential correlations in (C-13, N-15) labeled proteins: application to unfolded proteins. J Biomol NMR 20(2):135–147

    Article  Google Scholar 

  • Pannetier N, Houben K, Blanchard L, Marion D (2007) Optimized 3D-NMR sampling for resonance assignment of partially unfolded proteins. J Magn Reson 186(1):142–149

    Article  ADS  Google Scholar 

  • Perez Y, Gairi M, Pons M, Bernado 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(1):136–148

    Article  Google Scholar 

  • Pervushin K, Vogeli B, Eletsky A (2002) Longitudinal H-1 relaxation optimization in TROSY NMR spectroscopy. J Am Chem Soc 124(43):12898–12902

    Article  Google Scholar 

  • Peti W, Smith LJ, Redfield C, Schwalbe H (2001) Chemical shifts in denatured proteins: resonance assignments for denatured ubiquitin and comparisons with other denatured proteins. J Biomol NMR 19(2):153–165

    Article  Google Scholar 

  • Rovnyak D, Frueh DP, Sastry M, Sun ZYJ, 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(1):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 Mang Reson Spect 34(2):93–158

    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(22):8014–8015

    Article  Google Scholar 

  • Seepersaud R, Needham RHV, Kim CS, Jones AL (2006) Abundance of the δ subunit of RNA polymerase is linked to the virulence of Streptococcus agalactiae. J Bacteriol 188(8):2096–2105

    Article  Google Scholar 

  • Sklenář V (1995) Suppression of radiation damping in multidimensional NMR experiments using magentic-field gradients. J Magn Reson Ser A 114(1):132–135

    Article  Google Scholar 

  • Sørensen OW, Eich GW, Levitt MH, Bodenhausen G, Ernst RR (1984) Product operator formalism for the description of NMR pulse experiments. Prog Nucl Mang Reson Spect 16:163–192

    Article  Google Scholar 

  • Stern AS, Li KB, Hoch JC (2002) Modern spectrum analysis in multidimensional NMR spectroscopy: comparison of linear-prediction extrapolation and maximum-entropy reconstruction. J Am Chem Soc 124(9):1982–1993

    Article  Google Scholar 

  • Sun ZYJ, Frueh DP, Selenko P, Hoch JC, Wagner G (2005) Fast assignment of N-15-HSQC peaks using high-resolution 3D HNcocaNH experiments with non-uniform sampling. J Biomol NMR 33(1):43–50

    Article  Google Scholar 

  • Ward JJ, Sodhi JS, McGuffin LJ, Buxton BF, Jones DT (2004) Prediction and functional analysis of native disorder in proteins from the three kingdoms of life. J Mol Biol 337(3):635–645

    Article  Google Scholar 

  • Yao J, Chung J, Eliezer D, Wright PE, Dyson HJ (2001) NMR structural and dynamic characterization of the acid-unfolded state of apomyoglobin provides insights into the early events in protein folding. Biochemistry 40(12):3561–3571

    Article  Google Scholar 

  • 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(1):109–116

    Article  ADS  Google Scholar 

  • Zweckstetter M, Bax A (2001) Single-step determination of protein substructures using dipolar couplings: aid to structural genomics. J Am Chem Soc 123(39):9490–9491

    Article  Google Scholar 

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Acknowledgements

This work was supported by the Grants of the Ministry of Education of Czech Republic MSM0021622413 and LC06030, by the Grants 204/09/0583, 301/09/H004 and P206/11/0758 from Czech Science Foundation, by the EU/ grant POSTBIOMIN (FP7-REGPOT-2007-1 No. 205872), by MPD program from Foundation for Polish Sciences that was co-financed by the European Regional Development Fund. Financial support including the form of Access to the Bio-NMR Research Infrastructure co-funded under the 7th Framework Programme of the EC (FP7/2007-2013) grant agreement 261863 for conducting the research is gratefully acknowledged.

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Authors and Affiliations

  1. Faculty of Science, NCBR, and CEITEC, Masaryk University, Kamenice 5, 625 00, Brno, Czech Republic

    Jiří Nováček, Veronika Papoušková, Lukáš Žídek & Vladimír Sklenář

  2. Faculty of Chemistry, University of Warsaw, Pasteura 1, 02 093, Warsaw, Poland

    Anna Zawadzka-Kazimierczuk & Wiktor Koźmiński

  3. Laboratory of Molecular Genetics of Bacteria and Department of Bacteriology, Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídeňská 1083, 142 20, Prague, Czech Republic

    Hana Šanderová & Libor Krásný

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  1. Jiří Nováček
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  2. Anna Zawadzka-Kazimierczuk
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  3. Veronika Papoušková
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  4. Lukáš Žídek
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  8. Vladimír Sklenář
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Correspondence to Lukáš Žídek.

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Nováček, J., Zawadzka-Kazimierczuk, A., Papoušková, V. et al. 5D 13C-detected experiments for backbone assignment of unstructured proteins with a very low signal dispersion. J Biomol NMR 50, 1–11 (2011). https://doi.org/10.1007/s10858-011-9496-2

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  • DOI: https://doi.org/10.1007/s10858-011-9496-2

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