Journal of Biomolecular NMR

, Volume 62, Issue 3, pp 341–351 | Cite as

Fractional enrichment of proteins using [2-13C]-glycerol as the carbon source facilitates measurement of excited state 13Cα chemical shifts with improved sensitivity

  • Alexandra Ahlner
  • Cecilia Andresen
  • Shahid N. Khan
  • Lewis E. Kay
  • Patrik Lundström
Article
First Online:
Received:
Accepted:

Abstract

A selective isotope labeling scheme based on the utilization of [2-13C]-glycerol as the carbon source during protein overexpression has been evaluated for the measurement of excited state 13Cα chemical shifts using Carr–Purcell–Meiboom–Gill (CPMG) relaxation dispersion (RD) experiments. As expected, the fractional incorporation of label at the Cα positions is increased two-fold relative to labeling schemes based on [2-13C]-glucose, effectively doubling the sensitivity of NMR experiments. Applications to a binding reaction involving an SH3 domain from the protein Abp1p and a peptide from the protein Ark1p establish that accurate excited state 13Cα chemical shifts can be obtained from RD experiments, with errors on the order of 0.06 ppm for exchange rates ranging from 100 to 1000 s−1, despite the small fraction of 13Cα–13Cβ spin-pairs that are present for many residue types. The labeling approach described here should thus be attractive for studies of exchanging systems using 13Cα spin probes.

Keywords

CPMG 13Cα labeling [2-13C]-Glycerol Excited states 
This is a preview of subscription content, log in to check access.

Notes

Acknowledgments

We thank Dr. Martin Singull, Linköping University, for stimulating discussions and SWEDSTRUCT and the Swedish NMR Center for generous access to their high-field spectrometers. This work was supported by a grant from the Swedish Research Council (Dnr. 2012-5136) to P.L. L.E.K holds a Canadian Research Chair in Biochemistry.

Supplementary material

10858_2015_9948_MOESM1_ESM.docx (9.1 mb)
Supplementary material 1 (DOCX 9323 kb)

References

  1. Ahlner A, Carlsson M, Jonsson BH, Lundström P (2013) PINT—a software for integration of peak volumes and extraction of relaxation rates. J Biomol NMR 56:191–202CrossRefGoogle Scholar
  2. Auer R, Neudecker P, Muhandiram DR, Lundström P, Hansen DF, Konrat R, Kay LE (2009) Measuring the signs of 1Ha chemical shift differences between ground and excited protein states by off-resonance spin-lock R1r NMR spectroscopy. J Am Chem Soc 131:10832–10833CrossRefGoogle Scholar
  3. Bouvignies G, Vallurupalli P, Hansen DF, Correia BE, Lange O, Bah A, Vernon RM, Dahlquist FW, Baker D, Kay LE (2011) Solution structure of a minor and transiently formed state of a T4 lysozyme mutant. Nature 477:U111–U134ADSCrossRefGoogle Scholar
  4. Bouvignies G, Vallurupalli P, Kay LE (2014) Visualizing side chains of invisible protein conformers by solution NMR. J Mol Biol 426:763–774CrossRefGoogle Scholar
  5. Carr HY, Purcell EM (1954) Effects of diffusion on free precession in nuclear magnetic resonance experiments. Phys Rev 94:630–638ADSCrossRefGoogle Scholar
  6. Castellani F, van Rossum B, Diehl A, Schubert M, Rehbein K, Oschkinat H (2002) Structure of a protein determined by solid–state magic-angle-spinning NMR spectroscopy. Nature 420:98–102ADSCrossRefGoogle Scholar
  7. Cavalli A, Salvatella X, Dobson CM, Vendruscolo M (2007) Protein structure determination from NMR chemical shifts. Proc Natl Acad Sci USA 104:9615–9620ADSCrossRefGoogle Scholar
  8. Choy WY, Zhou Z, Bai YW, Kay LE (2005) An 15N NMR spin relaxation dispersion study of the folding of a pair of engineered mutants of apocytochrome b562. J Am Chem Soc 127:5066–5072CrossRefGoogle Scholar
  9. Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, Bax A (1995) NMRPipe—a multidimensional spectral processing system based on unix pipes. J Biomol NMR 6:277–293Google Scholar
  10. Dethoff EA, Petzold K, Chugh J, Casiano-Negroni A, Al-Hashimi HM (2012) Visualizing transient low-populated structures of RNA. Nature 491:724–728ADSGoogle Scholar
  11. Drubin DG, Mulholland J, Zhu ZM, Botstein D (1990) Homology of a yeast actin-binding protein to signal transduction proteins and myosin-I. Nature 343:288–290ADSCrossRefGoogle Scholar
  12. Forsén S, Hoffman RA (1963) Study of moderately rapid chemical exchange reactions by means of nuclear magnetic double resonance. J Chem Phys 39:2892–2901ADSCrossRefGoogle Scholar
  13. Geen H, Freeman R (1991) Band-selective radiofrequency pulses. J Magn Reson 93:93–141Google Scholar
  14. Hansen DF, Vallurupalli P, Lundström P, Neudecker P, Kay LE (2008) Probing chemical shifts of invisible states of proteins with relaxation dispersion NMR spectroscopy: how well can we do? J Am Chem Soc 130:2667–2675CrossRefGoogle Scholar
  15. Haynes J, Garcia B, Stollar EJ, Rath A, Andrews BJ, Davidson AR (2007) The biologically relevant targets and binding affinity requirements for the function of the yeast actin-binding protein 1 Src-homology 3 domain vary with genetic context. Genetics 176:193–208CrossRefGoogle Scholar
  16. Ishima R, Torchia DA (2003) Extending the range of amide proton relaxation dispersion experiments in proteins using a constant-time relaxation-compensated CPMG approach. J Biomol NMR 25:243–248CrossRefGoogle Scholar
  17. Ishima R, Baber J, Louis JM, Torchia DA (2004) Carbonyl carbon transverse relaxation dispersion measurements and ms–ms timescale motion in a protein hydrogen bond network. J Biomol NMR 29:187–198CrossRefGoogle Scholar
  18. Kimsey IJ, Petzold K, Sathyamoorthy B, Stein ZW, Al-Hashimi HM (2015) Visualizing transient Watson-Crick-like mispairs in DNA and RNA duplexes. Nature 519:315–320ADSCrossRefGoogle Scholar
  19. Korzhnev DM, Salvatella X, Vendruscolo M, Di Nardo AA, Davidson AR, Dobson CM, Kay LE (2004) Low-populated folding intermediates of Fyn SH3 characterized by relaxation dispersion NMR. Nature 430:586–590ADSCrossRefGoogle Scholar
  20. Korzhnev DM, Religa TL, Banachewicz W, Fersht AR, Kay LE (2010) A transient and low-populated protein-folding intermediate at atomic resolution. Science 329:1312–1316ADSCrossRefGoogle Scholar
  21. LeMaster DM, Kushlan DM (1996) Dynamical mapping of E. coli thioredoxin via 13C NMR relaxation analysis. J Am Chem Soc 118:9255–9264CrossRefGoogle Scholar
  22. Lila T, Drubin DG (1997) Evidence for physical and functional interactions among two Saccharomyces cerevisiae SH3 domain proteins, an adenylyl cyclase-associated protein and the actin cytoskeleton. Mol Biol Cell 8:367–385CrossRefGoogle Scholar
  23. Loria JP, Rance M, Palmer AG 3rd (1999) A relaxation-compensated Carr-Purcell-Meiboom-Gill sequence for characterizing chemical exchange by NMR spectroscopy. J Am Chem Soc 121:2331–2332CrossRefGoogle Scholar
  24. Lundström P, Teilum K, Carstensen T, Bezsonova I, Wiesner S, Hansen DF, Religa TL, Akke M, Kay LE (2007) Fractional 13C enrichment of isolated carbons using [1-13C]- or [2-13C]-glucose facilitates the accurate measurement of dynamics at backbone Ca and side-chain methyl positions in proteins. J Biomol NMR 38:199–212CrossRefGoogle Scholar
  25. Lundström P, Hansen DF, Kay LE (2008) Measurement of carbonyl chemical shifts of excited protein states by relaxation dispersion NMR spectroscopy: comparison between uniformly and selectively (13)C labeled samples. J Biomol NMR 42:35–47CrossRefGoogle Scholar
  26. Lundström P, Hansen DF, Vallurupalli P, Kay LE (2009a) Accurate measurement of alpha proton chemical shifts of excited protein states by relaxation dispersion NMR spectroscopy. J Am Chem Soc 131:1915–1926CrossRefGoogle Scholar
  27. Lundström P, Lin H, Kay LE (2009b) Measuring 13Cb chemical shifts of invisible excited states in proteins by relaxation dispersion NMR spectroscopy. J Biomol NMR 44:139–155CrossRefGoogle Scholar
  28. Lundström P, Vallurupalli P, Hansen DF, Kay LE (2009c) Isotope labeling methods for studies of excited protein states by relaxation dispersion NMR spectroscopy. Nat Protoc 4:1641–1648CrossRefGoogle Scholar
  29. Luz Z, Meiboom S (1963) Nuclear magnetic resonance study of the protolysis of trimethylammonium ion in aqueous solution-order of the reaction with respect to solvent. J Chem Phys 39:366–370ADSCrossRefGoogle Scholar
  30. Meiboom S, Gill D (1958) Modified spin-echo method for measuring nuclear relaxation times. Rev Sci Instrum 29:688–691ADSCrossRefGoogle Scholar
  31. Millet O, Loria JP, Kroenke CD, Pons M, Palmer AG 3rd (2000) The static magnetic field dependence of chemical exchange linebroadening defines the NMR chemical shift time scale. J Am Chem Soc 122:2867–2877CrossRefGoogle Scholar
  32. Mulder FAA, Skrynnikov NR, Hon B, Dahlquist FW, Kay LE (2001) Measurement of slow (ms-ms) time scale dynamics in protein side chains by 15N relaxation dispersion NMR spectroscopy: application to Asn and Gln residues in a cavity mutant of T4 lysozyme. J Am Chem Soc 123:967–975CrossRefGoogle Scholar
  33. Neudecker P, Robustelli P, Cavalli A, Walsh P, Lundström P, Zarrine-Afsar A, Sharpe S, Vendruscolo M, Kay LE (2012) Structure of an intermediate state in protein folding and aggregation. Science 336:362–366ADSCrossRefGoogle Scholar
  34. Palmer AG 3rd, Kroenke CD, Loria JP (2001) Nuclear magnetic resonance methods for quantifying microsecond-to-millisecond motions in biological macromolecules. Methods Enzymol 339:204–238CrossRefGoogle Scholar
  35. Rath A, Davidson AR (2000) The design of a hyperstable mutant of the Abp1p SH3 domain by sequence alignment analysis. Protein Sci 9:2457–2469CrossRefGoogle Scholar
  36. Santoro J, King GC (1992) A constant-time 2D Overbodenhausen experiment for inverse correlation of isotopically enriched species. J Magn Reson 97:202–207ADSGoogle Scholar
  37. Shen Y, Bax A (2010) SPARTA plus: a modest improvement in empirical NMR chemical shift prediction by means of an artificial neural network. J Biomol NMR 48:13–22CrossRefGoogle Scholar
  38. Shen Y, Lange O, Delaglio F, Rossi P, Aramini JM, Liu GH, Eletsky A, Wu YB, Singarapu KK, Lemak A, Ignatchenko A, Arrowsmith CH, Szyperski T, Montelione GT, Baker D, Bax A (2008) Consistent blind protein structure generation from NMR chemical shift data. Proc Natl Acad Sci USA 105:4685–4690ADSCrossRefGoogle Scholar
  39. Skrynnikov NR, Dahlquist FW, Kay LE (2002) Reconstructing NMR spectra of “invisible” excited protein states using HSQC and HMQC experiments. J Am Chem Soc 124:12352–12360CrossRefGoogle Scholar
  40. Vallurupalli P, Hansen DF, Stollar E, Meirovitch E, Kay LE (2007) Measurement of bond vector orientations in invisible excited states of proteins. Proc Natl Acad Sci USA 104:18473–18477ADSCrossRefGoogle Scholar
  41. Vallurupalli P, Hansen DF, Kay LE (2008) Structures of invisible, excited protein states by relaxation dispersion NMR spectroscopy. Proc Natl Acad Sci USA 105:11766–11771ADSCrossRefGoogle Scholar
  42. Vallurupalli P, Bouvignies G, Kay LE (2013) A computational study of the effects of 13C-13C scalar couplings on 13C CEST NMR spectra: towards studies on a uniformly 13C labeled protein. ChemBioChem 14:1709–1713CrossRefGoogle Scholar
  43. Voet D, Voet JG (1995) Biochemistry. Wiley, HobokenGoogle Scholar
  44. Vuister GW, Bax A (1992) Resolution enhancement and spectral editing of uniformly 13C enriched proteins by homonuclear broad band 13C decoupling. J Magn Reson 98:428–435ADSGoogle Scholar
  45. Wang YJ, Jardetzky O (2002) Probability-based protein secondary structure identification using combined NMR chemical-shift data. Protein Sci 11:852–861CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2015

Authors and Affiliations

  • Alexandra Ahlner
    • 1
  • Cecilia Andresen
    • 1
  • Shahid N. Khan
    • 1
  • Lewis E. Kay
    • 2
    • 3
  • Patrik Lundström
    • 1
  1. 1.Division of Chemistry, Department of Physics, Chemistry and BiologyLinköping UniversityLinköpingSweden
  2. 2.Departments of Molecular Genetics, Biochemistry and Chemistry, One King’s College CircleThe University of TorontoTorontoCanada
  3. 3.Program in Molecular Structure and Function, Hospital for Sick ChildrenTorontoCanada

Personalised recommendations