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Journal of Biomolecular NMR

, 42:35 | Cite as

Measurement of carbonyl chemical shifts of excited protein states by relaxation dispersion NMR spectroscopy: comparison between uniformly and selectively 13C labeled samples

  • Patrik Lundström
  • D. Flemming Hansen
  • Lewis E. Kay
Article

Abstract

Carr–Purcell–Meiboom–Gill (CPMG) relaxation dispersion nuclear magnetic resonance (NMR) spectroscopy has emerged as a powerful method for quantifying chemical shifts of excited protein states. For many applications of the technique that involve the measurement of relaxation rates of carbon magnetization it is necessary to prepare samples with isolated 13C spins so that experiments do not suffer from magnetization transfer between coupled carbon spins that would otherwise occur during the CPMG pulse train. In the case of 13CO experiments however the large separation between 13CO and 13Cα chemical shifts offers hope that robust 13CO dispersion profiles can be recorded on uniformly 13C labeled samples, leading to the extraction of accurate 13CO chemical shifts of the invisible, excited state. Here we compare such chemical shifts recorded on samples that are selectively labeled, prepared using [1-13C]-pyruvate and NaH13CO3, or uniformly labeled, generated from 13C-glucose. Very similar 13CO chemical shifts are obtained from analysis of CPMG experiments recorded on both samples, and comparison with chemical shifts measured using a second approach establishes that the shifts measured from relaxation dispersion are very accurate.

Keywords

Relaxation dispersion NMR Chemical exchange Carr–Purcell–Meiboom–Gill Excited protein states Chemical shifts 

Abbreviations

NMR

Nuclear magnetic resonance

CPMG

Carr–Purcell–Meiboom–Gill

Notes

Acknowledgments

We thank Dr. Elliott Stollar and Ms. Hong Lin for the gift of Ark1p peptide that was used in some of the experiments. This work was supported by a grant from the Canadian Institutes of Health Research (CIHR). P. L. and D. F. H. hold fellowships from the CIHR Training Grant on Protein Folding in Health and Disease (P. L.) and the CIHR (D. F. H.). The authors thank Dr. Pramodh Vallurupalli for useful discussions. L. E. K. is the recipient of a Canada Research Chair in Biochemistry.

References

  1. Ando I, Saito H, Tabeta R, Shoji A, Ozaki T (1984) Conformation dependent 13C NMR chemical shifts of poly(l-alanine) in the solid state—FPT INDO calculation of N-acetyl-N′-methyl-l-alanine amide as a model compound of poly(l-alanine). Macromolecules 17:457–461CrossRefADSGoogle Scholar
  2. Bax A (2003) Weak alignment offers new NMR opportunities to study protein structure and dynamics. Protein Sci 12:1–16CrossRefGoogle Scholar
  3. Boehr DD, McElheny D, Dyson HJ, Wright PE (2006) The dynamic energy landscape of dihydrofolate reductase catalysis. Science 313:1638–1642CrossRefADSGoogle Scholar
  4. Carr HY, Purcell EM (1954) Effects of diffusion on free precession in nuclear magnetic resonance experiments. Phys Rev 94:630–638CrossRefADSGoogle Scholar
  5. Cavalli A, Salvatella X, Dobson CM, Vendruscolo M (2007) Protein structure determination from NMR chemical shifts. Proc Natl Acad Sci USA 104:9615–9620CrossRefADSGoogle Scholar
  6. Cornilescu G, Delaglio F, Bax A (1999) Protein backbone angle restraints from searching a database for chemical shift and sequence homology. J Biomol NMR 13:289–302CrossRefGoogle Scholar
  7. 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 6:277–293CrossRefGoogle Scholar
  8. Drubin DG, Mulholland J, Zhu ZM, Botstein D (1990) Homology of a yeast actin-binding protein to signal transduction proteins and myosin-I. Nature 343:288–290CrossRefADSGoogle Scholar
  9. Eisenmesser EZ, Bosco DA, Akke M, Kern D (2002) Enzyme dynamics during catalysis. Science 295:1520–1523CrossRefADSGoogle Scholar
  10. Eisenmesser EZ, Millet O, Labeikovsky W, Korzhnev DM, Wolf-Watz M, Bosco DA, Skalicky JJ, Kay LE, Kern D (2005) Intrinsic dynamics of an enzyme underlies catalysis. Nature 438:117–121CrossRefADSGoogle Scholar
  11. Geen H, Freeman R (1991) Band-selective radiofrequency pulses. J Magn Reson 93:93–141Google Scholar
  12. Goddard TD, Kneller DG SPARKY 3, University of California, San FranciscoGoogle Scholar
  13. Gullion T, Baker DB, Conradi MS (1990) New, compensated Carr–Purcell sequences. J Magn Reson 89:479–484Google Scholar
  14. Hansen AF, Vallurupalli P, Kay LE (2008a) An improved 15N relaxation dispersion experiment for the measurement of millisecond time-scale dynamics in proteins. J Phys Chem B 112:5898–5904CrossRefGoogle Scholar
  15. Hansen DF, Vallurupalli P, Kay LE (2008b) Quantifying two-bond 1HN–13CO and one-bond 1Hα13Cα dipolar couplings of invisible protein states by spin-state selective relaxation dispersion NMR spectroscopy. J Am Chem Soc 130:8397–8405CrossRefGoogle Scholar
  16. Hansen DF, Vallurupalli P, Lundström P, Neudecker P, Kay LE (2008c) 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
  17. 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
  18. Hill RB, Bracken C, DeGrado WF, Palmer AG (2000) Molecular motions and protein folding: characterization of the backbone dynamics and folding equilibrium of α2D using 13C NMR spin relaxation. J Am Chem Soc 122:11610–11619CrossRefGoogle Scholar
  19. Hu JS, Bax A (1996) Measurement of three-bond 13C–13C J couplings between carbonyl and carbonyl/carboxyl carbons in isotopically enriched proteins. J Am Chem Soc 118:8170–8171CrossRefGoogle Scholar
  20. Ishima R, Louis JM, Torchia DA (2001) Optimized labeling of (CHD2)-13C methyl isotopomers in perdeuterated proteins: potential advantages for 13C relaxation studies of methyl dynamics of larger proteins. J Biomol NMR 21:167–171CrossRefGoogle Scholar
  21. Ishima R, Baber J, Louis JM, Torchia DA (2004) Carbonyl carbon transverse relaxation dispersion measurements and ms–μs timescale motion in a protein hydrogen bond network. J Biomol NMR 29:187–198CrossRefGoogle Scholar
  22. Kay LE, Ikura M, Tschudin R, Bax A (1990) 3-Dimensional triple-resonance NMR spectroscopy of isotopically enriched proteins. J Magn Reson 89:496–514Google Scholar
  23. Kay LE, Keifer P, Saarinen T (1992) Pure absorption gradient enhanced heteronuclear single quantum correlation spectroscopy with improved sensitivity. J Am Chem Soc 114:10663–10665CrossRefGoogle Scholar
  24. 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 430:586–590CrossRefADSGoogle Scholar
  25. Korzhnev DM, Religa TL, Lundström P, Fersht AR, Kay LE (2007) The folding pathway of an FF domain: characterization of an on-pathway intermediate state under folding conditions by 15N, 13Cα and 13C-methyl relaxation dispersion and 1H/2H-exchange NMR spectroscopy. J Mol Biol 372:497–512CrossRefGoogle Scholar
  26. Kupce E, Freeman R (1995) Adiabatic pulses for wide-band inversion and broad-band decoupling. J Magn Reson Ser A 115:273–276CrossRefGoogle Scholar
  27. Le HB, Oldfield E (1994) Correlation between 15N NMR chemical shifts in proteins and secondary structure. J Biomol NMR 4:341–348CrossRefGoogle Scholar
  28. Lee AL, Urbauer JL, Wand AJ (1997) Improved labeling strategy for 13C relaxation measurements of methyl groups in proteins. J Biomol NMR 9:437–440CrossRefGoogle Scholar
  29. LeMaster DM, Kushlan DM (1996) Dynamical mapping of E. coli thioredoxin via 13C NMR relaxation analysis. J Am Chem Soc 118:9255–9264CrossRefGoogle Scholar
  30. 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 8:367–385Google Scholar
  31. Loria JP, Rance M, Palmer AGIII (1999) A relaxation-compensated Carr–Purcell–Meiboom–Gill sequence for characterizing chemical exchange by NMR spectroscopy. J Am Chem Soc 121:2331–2332CrossRefGoogle Scholar
  32. 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 C-alpha and side-chain methyl positions in proteins. J Biomol NMR 38:199–212CrossRefGoogle Scholar
  33. 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 85:393–399Google Scholar
  34. McCoy MA, Mueller L (1992) Selective shaped pulse decoupling in NMR: homonuclear [13C] carbonyl decoupling. J Am Chem Soc 114:2108–2112CrossRefGoogle Scholar
  35. Meiboom S, Gill D (1958) Modified spin-echo method for measuring nuclear relaxation times. Rev Sci Instrum 29:688–691CrossRefADSGoogle Scholar
  36. Mihara H, Esaki N (2002) Bacterial cysteine desulfurases: their function and mechanisms. Appl Microbiol Biotechnol 60:12–23CrossRefGoogle Scholar
  37. Mulder FAA, Spronk CAEM, Slijper M, Kaptein R, Boelens R (1996) Improved HSQC experiments for the observation of exchange broadened signals. J Biomol NMR 8:223–228CrossRefGoogle Scholar
  38. Mulder FAA, Mittermaier A, Hon B, Dahlquist FW, Kay LE (2001) Studying excited states of proteins by NMR spectroscopy. Nat Struct Biol 8:932–935CrossRefGoogle Scholar
  39. Mulder FAA, Hon B, Mittermaier A, Dahlquist FW, Kay LE (2002) Slow internal dynamics in proteins: application of NMR relaxation dispersion spectroscopy to methyl groups in a cavity mutant of T4 lysozyme. J Am Chem Soc 124:1443–1451CrossRefGoogle Scholar
  40. Neal S, Nip AM, Zhang HY, Wishart DS (2003) Rapid and accurate calculation of protein 1H, 13C and 15N chemical shifts. J Biomol NMR 26:215–240CrossRefGoogle Scholar
  41. Palmer AGIII, Kroenke CD, Loria JP (2001) Nuclear magnetic resonance methods for quantifying microsecond-to-millisecond motions in biological macromolecules. Method Enzymol 339:204–238CrossRefGoogle Scholar
  42. Palmer AG, Grey MJ, Wang CY (2005) Solution NMR spin relaxation methods for characterizing chemical exchange in high-molecular-weight systems. Method Enzymol 394:430–465CrossRefGoogle Scholar
  43. Pardi A, Wagner G, Wuthrich K (1983) Protein conformation and proton NMR chemical shifts. Eur J Biochem 137:445–454CrossRefGoogle Scholar
  44. Prestegard JH, Mayer KL, Valafar H, Benison GC (2005) Determination of protein backbone structures from residual dipolar couplings. Method Enzymol 394:175–209CrossRefGoogle Scholar
  45. 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
  46. Schleucher J, Sattler M, Griesinger C (1993) Coherence selection by gradients without signal attenuation—application to the 3-dimensional HNCO experiment. Angew Chem Int Edit 32:1489–1491CrossRefGoogle Scholar
  47. Shaka AJ, Keeler J, Frenkiel T, Freeman R (1983) An improved sequence for broad-band decoupling—WALTZ-16. J Magn Reson 52:335–338Google Scholar
  48. Shen Y, Bax A (2007) Protein backbone chemical shifts predicted from searching a database for torsion angle and sequence homology. J Biomol NMR 38:289–302CrossRefGoogle Scholar
  49. 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
  50. Spera S, Bax A (1991) Empirical correlation between protein backbone conformation and Cα and Cβ 13C nuclear magnetic resonance chemical shifts. J Am Chem Soc 113:5490–5492CrossRefGoogle Scholar
  51. Sugase K, Dyson HJ, Wright PE (2007) Mechanism of coupled folding and binding of an intrinsically disordered protein. Nature 447:1021–1025CrossRefADSGoogle Scholar
  52. Teilum K, Brath U, Lundström P, Akke M (2006) Biosynthetic 13C labeling of aromatic side chains in proteins for NMR relaxation measurements. J Am Chem Soc 128:2506–2507CrossRefGoogle Scholar
  53. Tjandra N, Bax A (1997) Direct measurement of distances and angles in biomolecules by NMR in a dilute liquid crystalline medium. Science 278:1111–1114CrossRefADSGoogle Scholar
  54. Tollinger M, Skrynnikov NR, Mulder FAA, Forman-Kay JD, Kay LE (2001) Slow dynamics in folded and unfolded states of an SH3 domain. J Am Chem Soc 123:11341–11352CrossRefGoogle Scholar
  55. Tolman JR, Flanagan JM, Kennedy MA, Prestegard JH (1995) Nuclear magnetic dipole interactions in field-oriented proteins—information for structure determination in solution. Proc Natl Acad Sci USA 92:9279–9283CrossRefADSGoogle Scholar
  56. Vallurupalli P, Kay LE (2006) Complementarity of ensemble and single-molecule measures of protein motion: a relaxation dispersion NMR study of an enzyme complex. Proc Natl Acad Sci USA 103:11910–11915CrossRefADSGoogle Scholar
  57. 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 104:18473–18477CrossRefADSGoogle Scholar
  58. Vallurupalli P, Hansen DF, Kay LE (2008a) Probing structure in invisible protein states with anisotropic NMR chemical shifts. J Am Chem Soc 130:2734–2735CrossRefGoogle Scholar
  59. Vallurupalli P, Hansen DF, Kay LE (2008b) Structures of invisible, excited protein states by relaxation dispersion NMR spectroscopy. Proc Natl Acad Sci USA (in press)Google Scholar
  60. Voet D, Voet JG (1995) Biochemistry. Wiley, HobokenGoogle Scholar
  61. Vuister GW, Bax A (1992) Resolution enhancement and spectral editing of uniformly 13C enriched proteins by homonuclear broad band 13C decoupling. J Magn Reson 98:428–435Google Scholar
  62. Wagner G, Pardi A, Wuthrich K (1983) Hydrogen bond length and 1H NMR chemical shifts in proteins. J Am Chem Soc 105:5948–5949CrossRefGoogle Scholar
  63. Wand AJ, Bieber RJ, Urbauer JL, McEvoy RP, Gan ZH (1995) Carbon relaxation in randomly fractionally 13C-enriched proteins. J Magn Reson Ser B 108:173–175CrossRefGoogle Scholar
  64. Watt ED, Shimada H, Kovrigin EL, Loria JP (2007) The mechanism of rate-limiting motions in enzyme function. Proc Natl Acad Sci USA 104:11981–11986CrossRefADSGoogle Scholar
  65. Wishart DS, Case DA (2002) Use of chemical shifts in macromolecular structure determination. Method Enzymol 338:3–34CrossRefGoogle Scholar
  66. Wishart DS, Sykes BD (1994) The 13C chemical-shift index—a simple method for the identification of protein secondary structure using 13C chemical-shift data. J Biomol NMR 4:171–180CrossRefGoogle Scholar
  67. Wishart DS, Sykes BD, Richards FM (1991) Relationship between nuclear magnetic resonance chemical shift and protein secondary structure. J Mol Biol 222:311–333CrossRefGoogle Scholar
  68. Wolf-Watz M, Thai V, Henzler-Wildman K, Hadjipavlou G, Eisenmesser EZ, Kern D (2004) Linkage between dynamics and catalysis in a thermophilic-mesophilic enzyme pair. Nat Struct Mol Biol 11:945–949CrossRefGoogle Scholar
  69. Xu XP, Case DA (2002) Probing multiple effects on 15N, 13Cα, 13Cβ, and 13C′ chemical shifts in peptides using density functional theory. Biopolymers 65:408–423CrossRefGoogle Scholar
  70. Ying JF, Chill JH, Louis JM, Bax A (2007) Mixed-time parallel evolution in multiple quantum NMR experiments: sensitivity and resolution enhancement in heteronuclear NMR. J Biomol NMR 37:195–204CrossRefGoogle Scholar
  71. Zeeb M, Balbach J (2005) NMR spectroscopic characterization of millisecond protein folding by transverse relaxation dispersion measurements. J Am Chem Soc 127:13207–13212CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2008

Authors and Affiliations

  • Patrik Lundström
    • 1
    • 2
    • 3
  • D. Flemming Hansen
    • 1
    • 2
    • 3
  • Lewis E. Kay
    • 1
    • 2
    • 3
  1. 1.Department of Medical GeneticsThe University of TorontoTorontoCanada
  2. 2.Department of BiochemistryThe University of TorontoTorontoCanada
  3. 3.Department of ChemistryThe University of TorontoTorontoCanada

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