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Speeding up sequence specific assignment of IDPs

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Abstract

The characterization of intrinsically disordered proteins (IDPs) by NMR spectroscopy is made difficult by the extensive spectral overlaps. To overcome the intrinsic low-resolution of the spectra the introduction of high-dimensionality experiments is essential. We present here a set of high-resolution experiments based on direct 13C-detection which proved useful in the assignment of α-synuclein, a paradigmatic IDP. In particular, we describe the implementation of 4D HCBCACON, HCCCON, HCBCANCO, 4/5D HNCACON and HNCANCO and 3/4D HCANCACO experiments, specifically tailored for spin system identification and backbone resonances sequential assignment. The use of non-uniform-sampling in the indirect dimension and of the H-flip approach to achieve longitudinal relaxation enhancement rendered the experiments very practical.

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References

  • Atreya HS, Szyperski T (2005) Rapid NMR data collection. Methods Enzymol 394:78–108

    Article  Google Scholar 

  • Bermel W, Bertini I, Felli IC, Kümmerle R, Pierattelli R (2003) 13C direct detection experiments on the paramagnetic oxidized monomeric copper, zinc superoxide dismutase. J Am Chem Soc 125:16423–16429

    Article  Google Scholar 

  • 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 178:56–64

    Article  ADS  Google Scholar 

  • 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 128:3918–3919

    Article  Google Scholar 

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

    Article  Google Scholar 

  • Bermel W, Felli IC, Kümmerle R, Pierattelli R (2008) 13C direct-detection biomolecular NMR. Concepts Magn Reson 32A:183–200

    Article  Google Scholar 

  • 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 198:275–281

    Article  ADS  Google Scholar 

  • Bermel W, Bertini I, Felli IC, Pierattelli R (2009b) Speeding up 13C direct detection NMR experiments. J Am Chem Soc 131:15339–15345

    Article  Google Scholar 

  • Bermel W, Bertini I, Felli IC, Peruzzini R, Pierattelli R (2010) Exclusively heteronuclear NMR experiments to obtain structural and dynamic information on proteins. ChemPhysChem 11:689–695

    Article  Google Scholar 

  • Bertini I, Felli IC, Gonnelli L, Vasantha Kumar MV, Pierattelli R (2011a) 13C direct-detection biomolecular NMR spectroscopy in living cells. Angew Chem Int Ed 50:1–4

    Article  Google Scholar 

  • Bertini I, Felli IC, Gonnelli L, Vasantha Kumar MV, Pierattelli R (2011b) High-resolution characterization of intrinsic disorder in proteins. ChemBioChem 12:2347–2352

    Article  Google Scholar 

  • Boehlen J-M, Bodenhausen G (1993) Experimental aspects of chirp NMR spectroscopy. J Magn Reson, Ser A 102:293–301

    Article  Google Scholar 

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

    Article  Google Scholar 

  • Deschamps M, Campbell ID (2006) Cooling overall spin temperature: protein NMR experiments optimized for longitudinal relaxation effects. J Magn Reson 178:206–211

    Article  ADS  Google Scholar 

  • Dunker AK, Lawson JD, Brown CJ, Williams RM, Romero P, Oh JS, Ratliff CM, Hipps KW, Ausio J, Nissen MS, Reeves R, Kang C, Kissinger CR, Bailey RW, Griswold MD, Chiu W, Garner EC (2001) Intrinsically disordered protein. J Mol Graph Model 19:26–59

    Article  Google Scholar 

  • Dyson HJ, Wright PE (2004) Unfolded proteins and protein folding studied by NMR. Chem Rev 104:3607–3622

    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 

  • Eliezer D (2009) Biophysical characterization of intrinsically disordered proteins. Curr Opin Struct Biol 19:23–30

    Article  Google Scholar 

  • Emsley L, Bodenhausen G (1992) Optimization of shaped selective pulses for NMR using a quaternion description of their overall propagators. J Magn Reson 97:135–148

    Google Scholar 

  • Felli IC, Pierattelli R (2012) 13C direct detection NMR. In: McGreevy KS, Parigi G (eds) Bertini I, NMR of biomolecules. Wiley-Blackwell, London, pp 433–442

    Google Scholar 

  • Goddard TD, Kneller DG (2000) SPARKY 3. University of Califofnia, San Francisco

    Google Scholar 

  • Hiller S, Fiorito F, Wüthrich K, Wider G (2005) Automated projection spectroscopy (APSY). Proc Natl Acad Sci USA 102:10876–10881

    Article  ADS  Google Scholar 

  • 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 131:7222–7223

    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 

  • 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 42:173–177

    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 

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

    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 188:344–356

    Article  ADS  Google Scholar 

  • Kazimierczuk K, Zawadzka A, Koźmiński W (2008) Optimization of random time domain sampling in multidimensional NMR. J Magn Reson 192:123–130

    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 197:219–228

    Article  ADS  Google Scholar 

  • Kazimierczuk K, Stanek J, Zawadzka-Kazimierczuk A, Koźmiński W (2010) Random sampling in multidimensional NMR spectroscopy. Prog NMR Spectrosc 57:420–434

    Article  Google Scholar 

  • Kazimierczuk K, Misiak M, Stanek J, Zawadzka-Kazimierczuk A, Koźmiński W (2012) Generalized Fourier transform for non-uniform sampled data. Top Curr Chem 316:79–124

    Article  Google Scholar 

  • Keller RLJ (2004) The computer aided resonance assignment tutorial. Cantina Verag, Goldau

    Google Scholar 

  • Kjaergaard M, Poulsen FM (2011) Sequence correction of random coil chemical shifts: correlation between neighbor correction factors and changes in the Ramachandran distribution. J Biomol NMR 50:157–165

    Article  Google Scholar 

  • Kjaergaard M, Brander S, Poulsen FM (2011) Random coil chemical shift for intrinsically disordered proteins: effects of temperature and pH. J Biomol NMR 49:139–149

    Article  Google Scholar 

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

    Article  Google Scholar 

  • Lescop E, Schanda P, Brutscher B (2007) A set of BEST triple resonance experiments for time-optimized protein resonance assignment. J Magn Reson 187:163–169

    Article  ADS  Google Scholar 

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

    Article  Google Scholar 

  • Mittag T, Forman-Kay J (2007) Atomic-level characterization of disordered protein ensembles. Curr Opin Struct Biol 17:3–14

    Article  Google Scholar 

  • 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 132:11906–11907

    Article  Google Scholar 

  • Nováček J, Zawadzka-Kazimierczuk A, Papoušková V, Žídek L, Šanderová H, Krásny 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 50:1–11

    Article  Google Scholar 

  • 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 200:354–358

    Article  ADS  Google Scholar 

  • Pasat G, Zintsmaster JS, Peng J (2008) Direct 13C-detection for carbonyl relaxation studies of protein dynamics. J Magn Reson 193:226–232

    Article  ADS  Google Scholar 

  • Pérez Y, Gairi M, Pons M, Bernadò 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 391:136–148

    Article  Google Scholar 

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

    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 127:8014–8015

    Article  Google Scholar 

  • Schanda P, Van Melckebeke H, Brutscher B (2006) Speeding up three-dimensional protein NMR experiments to a few minutes. J Am Chem Soc 128:9042–9043

    Article  Google Scholar 

  • Serber Z, Richter C, Moskau D, Boehlen J-M, Gerfin T, Marek D, Haeberli M, Baselgia L, Laukien F, Stern AS, Hoch JC, Dötsch V (2000) New carbon-detected protein NMR experiments using cryoprobes. J Am Chem Soc 122:3554–3555

    Article  Google Scholar 

  • Shaka AJ, Keeler J, Freeman R (1983) Evaluation of a new broadband decoupling sequence: WALTZ-16. J Magn Reson 53:313–340

    Google Scholar 

  • Shaka AJ, Barker PB, Freeman R (1985) Computer-optimized decoupling scheme for wideband applications and low-level operation. J Magn Reson 64:547–552

    Google Scholar 

  • Shimba N, Kovacs H, Stern AS, Nomura AM, Shimada I, Hoch JC, Craik CS, Dötsch V (2005) Optimization of 13C direct detection NMR methods. J Biomol NMR 30:175–179

    Article  Google Scholar 

  • Stanek J, Augustyniak R, Koźmiński W (2012) Suppression of sampling artefacts in high-resolution four-dimensional NMR spectra using signal separation algorithm. J Magn Reson 214:91–102

    Article  ADS  Google Scholar 

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

    Article  Google Scholar 

  • Tompa P (2009) Structure and function of intrinsically disordered proteins. Taylor and Francis Group, Boca Raton

    Book  Google Scholar 

  • Uversky VN, Gillespie JR, Fink AL (2000) Why are “natively unfolded” proteins unstructured under physiologic conditions? Proteins Struct Funct Genet 41:415–427

    Article  Google Scholar 

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

    Article  Google Scholar 

  • Zawadzka-Kazimierczuk A, Koźmiński W, Šanderová H, Krásny L (2012) High dimensional and high resolution pulse sequences for backbone resonance assignment of intrinsically disordered proteins. J Biomol NMR 52:329–337

    Article  Google Scholar 

  • Zhang H, Neal S, Wishart DS (2003) RefDB: a database of uniformly referenced protein chemical shifts. J Biomol NMR 25:173–195

    Article  Google Scholar 

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Acknowledgments

This work has been supported in part by the EC 7th Framework program BioNMR, contract 261863 and by the EC Marie Curie ITN program (IDPbyNMR, contract 264257).

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Authors and Affiliations

  1. Bruker BioSpin GmbH, Silberstreifen, 76287, Rheinstetten, Germany

    Wolfgang Bermel

  2. CERM, University of Florence, Via Luigi Sacconi 6, 50019, Sesto Fiorentino, Florence, Italy

    Ivano Bertini, Isabella C. Felli, Leonardo Gonnelli, Alessandro Piai & Roberta Pierattelli

  3. Department of Chemistry “Ugo Schiff”, University of Florence, 50019, Sesto Fiorentino, Italy

    Ivano Bertini, Isabella C. Felli & Roberta Pierattelli

  4. Faculty of Chemistry, University of Warsaw, Pasteura 1, 02093, Warsaw, Poland

    Wiktor Koźmiński & Jan Stanek

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  1. Wolfgang Bermel
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Correspondence to Ivano Bertini.

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Bermel, W., Bertini, I., Felli, I.C. et al. Speeding up sequence specific assignment of IDPs. J Biomol NMR 53, 293–301 (2012). https://doi.org/10.1007/s10858-012-9639-0

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