Journal of Biomolecular NMR

, Volume 45, Issue 1–2, pp 45–55 | Cite as

CPMG relaxation dispersion NMR experiments measuring glycine 1Hα and 13Cα chemical shifts in the ‘invisible’ excited states of proteins

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

Abstract

Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion NMR experiments are extremely powerful for characterizing millisecond time-scale conformational exchange processes in biomolecules. A large number of such CPMG experiments have now emerged for measuring protein backbone chemical shifts of sparsely populated (>0.5%), excited state conformers that cannot be directly detected in NMR spectra and that are invisible to most other biophysical methods as well. A notable deficiency is, however, the absence of CPMG experiments for measurement of 1Hα and 13Cα chemical shifts of glycine residues in the excited state that reflects the fact that in this case the 1Hα, 13Cα spins form a three-spin system that is more complex than the AX 1Hα13Cα spin systems in the other amino acids. Here pulse sequences for recording 1Hα and 13Cα CPMG relaxation dispersion profiles derived from glycine residues are presented that provide information from which 1Hα, 13Cα chemical shifts can be obtained. The utility of these experiments is demonstrated by an application to a mutant of T4 lysozyme that undergoes a millisecond time-scale exchange process facilitating the binding of hydrophobic ligands to an internal cavity in the protein.

Keywords

CPMG Relaxation dispersion Excited protein states T4 lysozyme Millisecond dynamics 

Notes

Acknowledgments

This work was supported by funds from the Canadian Institutes of Health Research (CIHR) in the form of a research grant to LEK and postdoctoral fellowships to DFH and PL (Protein Folding Training Grant). LEK holds a Canada Research Chair in Biochemistry.

Supplementary material

10858_2009_9310_MOESM1_ESM.pdf (279 kb)
(PDF 279 kb)

References

  1. Austin RH, Beeson KW, Eisenstein L, Frauenfelder H, Gunsalus IC (1975) Dynamics of ligand binding to myoglobin. Biochemistry 14:5355–5373CrossRefGoogle Scholar
  2. Bax A, Ikura M, Kay LE, Torchia DA, Tschudin R (1990) Comparison of different modes of 2-dimensional reverse-correlation NMR for the study of proteins. J Magn Reson 86:304–318Google 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. Burum DP, Ernst RR (1980) Net polarization transfer via a J-ordered state for signal enhancement of low-sensitivity nuclei. J Magn Reson 39:163–168Google Scholar
  5. Carr HY, Purcell EM (1954) Effects of diffusion on free precession in nuclear magnetic resonance experiments. Phys Rev 94:630–638CrossRefADSGoogle 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. Eriksson AE, Baase WA, Wozniak JA, Matthews BW (1992) A cavity-containing mutant of T4 lysozyme is stabilized by buried benzene. Nature 355:371–373CrossRefADSGoogle Scholar
  9. Feher VA, Baldwin EP, Dahlquist FW (1996) Access of ligands to cavities within the core of a protein is rapid. Nat Struct Biol 3:516–521CrossRefGoogle Scholar
  10. Geen H, Freeman R (1991) Band-selective radiofrequency pulses. J Magn Reson 93:93–141Google Scholar
  11. Goddard TD, Kneller DG. SPARKY 3. University of California, San FranciscoGoogle Scholar
  12. Grey MJ, Wang C, Palmer AG III (2003) Disulfide bond isomerization in basic pancreatic trypsin inhibitor: multisite chemical exchange quantified by CPMG relaxation dispersion and chemical shift modeling. J Am Chem Soc 125:14324–14335CrossRefGoogle Scholar
  13. Grey MJ, Tang Y, Alexov E, McKnight CJ, Raleigh DP, Palmer AG III (2006) Characterizing a partially folded intermediate of the villin headpiece domain under non-denaturing conditions: contribution of His41 to the pH-dependent stability of the N-terminal subdomain. J Mol Biol 355:1078–1094CrossRefGoogle Scholar
  14. Griffey RH, Redfield AG (1987) Proton-detected heteronuclear edited and correlated nuclear magnetic resonance and nuclear overhauser effect in solution. Q Rev Biophys 19:51–82CrossRefGoogle Scholar
  15. Grzesiek S, Bax A (1995) Spin-locked multiple quantum coherence for signal enhancement in heteronuclear multidimensional NMR experiments. J Biomol NMR 6:335–339CrossRefGoogle Scholar
  16. Hansen PE (2000) Isotope effects on chemical shifts of proteins and peptides. Magn Reson Chem 38:1–10CrossRefGoogle Scholar
  17. Hansen DF, 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
  18. Hansen DF, Vallurupalli P, Kay LE (2008b) Quantifying two-bond 1HN-13CO and one-bond 1H(alpha)-13C(alpha) dipolar couplings of invisible protein states by spin-state selective relaxation dispersion NMR spectroscopy. J Am Chem Soc 130:8397–8405CrossRefGoogle Scholar
  19. Hansen DF, Vallurupalli P, Kay LE (2008c) Using relaxation dispersion NMR spectroscopy to determine structures of excited, invisible protein states. J Biomol NMR 41:113–120CrossRefGoogle Scholar
  20. Hansen DF, Vallurupalli P, Lundstrom P, Neudecker P, Kay LE (2008d) 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
  21. Henzler-Wildman KA, Thai V, Lei M, Ott M, Wolf-Watz M, Fenn T, Pozharski E, Wilson MA, Petsko GA, Karplus M, Hubner CG, Kern D (2007) Intrinsic motions along an enzymatic reaction trajectory. Nature 450:838–844CrossRefADSGoogle Scholar
  22. Igumenova TI, Brath U, Akke M, Palmer AG III (2007) Characterization of chemical exchange using residual dipolar coupling. J Am Chem Soc 129:13396–13397CrossRefGoogle Scholar
  23. 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
  24. Ishima R, Wingfield PT, Stahl SJ, Kaufman JD, Torchia DA (1998) Using amide H-1 and N-15 transverse relaxation to detect millisecond time-scale motions in perdeuterated proteins: application to HIV-1 protease. J Am Chem Soc 120:10534–10542CrossRefGoogle Scholar
  25. Karplus M (2000) Aspects of protein reaction dynamics: deviations from simple behavior. J Phys Chem B 104:11–27CrossRefGoogle Scholar
  26. Karplus M, Kuriyan J (2005) Molecular dynamics and protein function. Proc Natl Acad Sci USA 102:6679–6685CrossRefADSGoogle Scholar
  27. Korzhnev DM, Kay LE (2008) Probing invisible, low-populated states of protein molecules by relaxation dispersion NMR spectroscopy: an application to protein folding. Acc Chem Res 41:442–451CrossRefGoogle Scholar
  28. 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
  29. Kupce E, Freeman R (1996) Optimized adiabatic pulses for wideband spin inversion. J Magn Reson A 118:299–303CrossRefGoogle Scholar
  30. Kushlan DM, Lemaster DM (1993) Resolution and sensitivity enhancement of heteronuclear correlation for methylene resonances via H-2-enrichment and decoupling. J Biomol NMR 3:701–708CrossRefGoogle Scholar
  31. Loria JP, Rance M, Palmer AG (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. Lundstrom 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
  33. Lundstrom P, Hansen DF, Vallurupalli P, Kay LE (2009) Accurate measurement of alpha proton chemical shifts of excited protein states by relaxation dispersion NMR spectroscopy. J Am Chem Soc 131:1915–1926CrossRefGoogle Scholar
  34. 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
  35. Meiboom S, Gill D (1958) Modified spin-echo method for measuring nuclear relaxation times. Rev Sci Instrum 29:688–691CrossRefADSGoogle Scholar
  36. Miclet E, Williams DC, Clore GM, Bryce DL, Boisbouvier J, Bax A (2004) Relaxation-optimized NMR spectroscopy of methylene groups in proteins and nucleic acids. J Am Chem Soc 126:10560–10570CrossRefGoogle Scholar
  37. Mittermaier A, Kay LE (2006) New tools provide new insights in NMR studies of protein dynamics. Science 312:224–228CrossRefADSGoogle Scholar
  38. Mulder FA, Hon B, Muhandiram DR, Dahlquist FW, Kay LE (2000) Flexibility and ligand exchange in a buried cavity mutant of T4 lysozyme studied by multinuclear NMR. Biochemistry 39:12614–12622CrossRefGoogle Scholar
  39. Mulder FA, Mittermaier A, Hon B, Dahlquist FW, Kay LE (2001a) Studying excited states of proteins by NMR spectroscopy. Nat Struct Biol 8:932–935CrossRefGoogle Scholar
  40. Mulder FA, Skrynnikov NR, Hon B, Dahlquist FW, Kay LE (2001b) Measurement of slow (micros-ms) time scale dynamics in protein side chains by (15)N 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
  41. Palmer AG (2004) NMR characterization of the dynamics of biomacromolecules. Chem Rev 104:3623–3640CrossRefGoogle Scholar
  42. Palmer AG, Cavanagh J, Wright PE, Rance M (1991) Sensitivity improvement in proton-detected 2-dimensional heteronuclear correlation Nmr-spectroscopy. J Magn Reson 93:151–170Google Scholar
  43. Palmer AG III, Kroenke CD, Loria JP (2001) Nuclear magnetic resonance methods for quantifying microsecond-to-millisecond motions in biological macromolecules. Methods Enzymol 339:204–238CrossRefGoogle Scholar
  44. Ramakrishnan C, Ramachandran GN (1965) Stereochemical criteria for polypeptide and protein chain conformations.2. Allowed conformations for a pair of peptide units. Biophys J 5:909–933CrossRefGoogle Scholar
  45. Roy S, Papastavros MZ, Sanchez V, Redfield AG (1984) Nitrogen-15-labeled yeast tRNAPhe: double and two-dimensional heteronuclear NMR of guanosine and uracil ring NH groups. Biochemistry 23:4395–4400CrossRefGoogle Scholar
  46. 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
  47. 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
  48. States DJ, Haberkorn RA, Ruben DJ (1982) A two-dimensional nuclear overhauser experiment with pure absorption phase in 4 quadrants. J Magn Reson 48:286–292Google Scholar
  49. Sugase K, Dyson HJ, Wright PE (2007) Mechanism of coupled folding and binding of an intrinsically disordered protein. Nature 447:1021–1025CrossRefADSGoogle Scholar
  50. 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
  51. Vallurupalli P, Hansen DF, Stollar E, Meirovitch E, Kay LE (2007a) Measurement of bond vector orientations in invisible excited states of proteins. Proc Natl Acad Sci USA 104:18473–18477CrossRefADSGoogle Scholar
  52. Vallurupalli P, Scott L, Williamson JR, Kay LE (2007b) Strong coupling effects during X-pulse CPMG experiments recorded on heteronuclear ABX spin systems: artifacts and a simple solution. J Biomol NMR 38:41–46CrossRefGoogle Scholar
  53. 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
  54. Vallurupalli P, Hansen DF, Kay LE (2008b) Structures of invisible, excited protein states by relaxation dispersion NMR spectroscopy. Proc Natl Acad Sci USA 105:11766–11771CrossRefADSGoogle Scholar
  55. 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

Copyright information

© Springer Science+Business Media B.V. 2009

Authors and Affiliations

  • Pramodh Vallurupalli
    • 1
  • D. Flemming Hansen
    • 1
  • Patrik Lundström
    • 1
  • Lewis E. Kay
    • 1
  1. 1.Departments of Molecular Genetics, Biochemistry and ChemistryUniversity of TorontoTorontoCanada

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