Skip to main content
SpringerLink
Log in

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

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

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.

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

  • 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 56:191–202

    Article  Google Scholar 

  • 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 131:10832–10833

    Article  Google Scholar 

  • 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–U134

    Article  ADS  Google Scholar 

  • Bouvignies G, Vallurupalli P, Kay LE (2014) Visualizing side chains of invisible protein conformers by solution NMR. J Mol Biol 426:763–774

    Article  Google Scholar 

  • Carr HY, Purcell EM (1954) Effects of diffusion on free precession in nuclear magnetic resonance experiments. Phys Rev 94:630–638

    Article  ADS  Google Scholar 

  • 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 420:98–102

    Article  ADS  Google Scholar 

  • Cavalli A, Salvatella X, Dobson CM, Vendruscolo M (2007) Protein structure determination from NMR chemical shifts. Proc Natl Acad Sci USA 104:9615–9620

    Article  ADS  Google Scholar 

  • 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 127:5066–5072

    Article  Google Scholar 

  • 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 6:277–293

    Google Scholar 

  • Dethoff EA, Petzold K, Chugh J, Casiano-Negroni A, Al-Hashimi HM (2012) Visualizing transient low-populated structures of RNA. Nature 491:724–728

    ADS  Google Scholar 

  • Drubin DG, Mulholland J, Zhu ZM, Botstein D (1990) Homology of a yeast actin-binding protein to signal transduction proteins and myosin-I. Nature 343:288–290

    Article  ADS  Google Scholar 

  • Forsén S, Hoffman RA (1963) Study of moderately rapid chemical exchange reactions by means of nuclear magnetic double resonance. J Chem Phys 39:2892–2901

    Article  ADS  Google Scholar 

  • Geen H, Freeman R (1991) Band-selective radiofrequency pulses. J Magn Reson 93:93–141

  • 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 130:2667–2675

    Article  Google Scholar 

  • 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 176:193–208

    Article  Google Scholar 

  • 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 25:243–248

    Article  Google Scholar 

  • 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 29:187–198

    Article  Google Scholar 

  • Kimsey IJ, Petzold K, Sathyamoorthy B, Stein ZW, Al-Hashimi HM (2015) Visualizing transient Watson-Crick-like mispairs in DNA and RNA duplexes. Nature 519:315–320

    Article  ADS  Google Scholar 

  • 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 430:586–590

    Article  ADS  Google Scholar 

  • Korzhnev DM, Religa TL, Banachewicz W, Fersht AR, Kay LE (2010) A transient and low-populated protein-folding intermediate at atomic resolution. Science 329:1312–1316

    Article  ADS  Google Scholar 

  • LeMaster DM, Kushlan DM (1996) Dynamical mapping of E. coli thioredoxin via 13C NMR relaxation analysis. J Am Chem Soc 118:9255–9264

    Article  Google Scholar 

  • 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 8:367–385

    Article  Google Scholar 

  • 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 121:2331–2332

    Article  Google Scholar 

  • 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 38:199–212

    Article  Google Scholar 

  • 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 42:35–47

    Article  Google Scholar 

  • 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 131:1915–1926

    Article  Google Scholar 

  • 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 44:139–155

    Article  Google Scholar 

  • 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 4:1641–1648

    Article  Google Scholar 

  • 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 39:366–370

    Article  ADS  Google Scholar 

  • Meiboom S, Gill D (1958) Modified spin-echo method for measuring nuclear relaxation times. Rev Sci Instrum 29:688–691

    Article  ADS  Google Scholar 

  • 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 122:2867–2877

    Article  Google Scholar 

  • 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 123:967–975

    Article  Google Scholar 

  • 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 336:362–366

    Article  ADS  Google Scholar 

  • Palmer AG 3rd, Kroenke CD, Loria JP (2001) Nuclear magnetic resonance methods for quantifying microsecond-to-millisecond motions in biological macromolecules. Methods Enzymol 339:204–238

    Article  Google Scholar 

  • Rath A, Davidson AR (2000) The design of a hyperstable mutant of the Abp1p SH3 domain by sequence alignment analysis. Protein Sci 9:2457–2469

    Article  Google Scholar 

  • Santoro J, King GC (1992) A constant-time 2D Overbodenhausen experiment for inverse correlation of isotopically enriched species. J Magn Reson 97:202–207

    ADS  Google Scholar 

  • 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 48:13–22

    Article  Google Scholar 

  • 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 105:4685–4690

    Article  ADS  Google Scholar 

  • Skrynnikov NR, Dahlquist FW, Kay LE (2002) Reconstructing NMR spectra of “invisible” excited protein states using HSQC and HMQC experiments. J Am Chem Soc 124:12352–12360

    Article  Google Scholar 

  • 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 104:18473–18477

    Article  ADS  Google Scholar 

  • Vallurupalli P, Hansen DF, Kay LE (2008) Structures of invisible, excited protein states by relaxation dispersion NMR spectroscopy. Proc Natl Acad Sci USA 105:11766–11771

    Article  ADS  Google Scholar 

  • 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 14:1709–1713

    Article  Google Scholar 

  • Voet D, Voet JG (1995) Biochemistry. Wiley, Hoboken

  • Vuister GW, Bax A (1992) Resolution enhancement and spectral editing of uniformly 13C enriched proteins by homonuclear broad band 13C decoupling. J Magn Reson 98:428–435

    ADS  Google Scholar 

  • Wang YJ, Jardetzky O (2002) Probability-based protein secondary structure identification using combined NMR chemical-shift data. Protein Sci 11:852–861

    Article  Google Scholar 

Download references

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.

Author information

Authors and Affiliations

  1. Division of Chemistry, Department of Physics, Chemistry and Biology, Linköping University, 58183, Linköping, Sweden

    Alexandra Ahlner, Cecilia Andresen, Shahid N. Khan & Patrik Lundström

  2. Departments of Molecular Genetics, Biochemistry and Chemistry, One King’s College Circle, The University of Toronto, Toronto, ON, M5S 1A8, Canada

    Lewis E. Kay

  3. Program in Molecular Structure and Function, Hospital for Sick Children, 555 University Avenue, Toronto, ON, M5G 1X8, Canada

    Lewis E. Kay

Authors
  1. Alexandra Ahlner
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Cecilia Andresen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Shahid N. Khan
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Lewis E. Kay
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Patrik Lundström
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Patrik Lundström.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 9323 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ahlner, A., Andresen, C., Khan, S.N. et al. Fractional enrichment of proteins using [2-13C]-glycerol as the carbon source facilitates measurement of excited state 13Cα chemical shifts with improved sensitivity. J Biomol NMR 62, 341–351 (2015). https://doi.org/10.1007/s10858-015-9948-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10858-015-9948-1

Keywords

Search

Navigation