Skip to main content
SpringerLink
Log in

Analyzing Protein–Ligand Interactions by Dynamic NMR Spectroscopy

  • Protocol
  • First Online:
Protein-Ligand Interactions

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1008))

Abstract

Nuclear magnetic resonance (NMR) spectroscopy can provide detailed information on protein–ligand interactions that is inaccessible using other biophysical techniques. This chapter focuses on NMR-based approaches for extracting affinity and rate constants for weakly binding transient protein complexes with lifetimes of less than about a second. Several pulse sequences and analytical techniques are discussed, including line-shape simulations, spin-echo relaxation dispersion methods (CPMG), and magnetization exchange (EXSY) experiments.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 139.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 179.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 219.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Similar content being viewed by others

References

  1. Cavanagh J (1996) Protein NMR spectroscopy: principles and practice. Academic, San Diego

    Google Scholar 

  2. Mittermaier A, Kay LE (2006) New tools provide new insights in NMR studies of protein dynamics. Science 312:224–228

    Article  PubMed  CAS  Google Scholar 

  3. Ikura M, Clore GM, Gronenborn AM, Zhu G, Klee CB, Bax A (1992) Solution structure of a calmodulin-target peptide complex by multidimensional NMR. Science 256:632–638

    Article  PubMed  CAS  Google Scholar 

  4. Walters KJ, Ferentz AE, Hare BJ, Hidalgo P, Jasanoff A, Matsuo H, Wagner G (2001) Characterizing protein–protein complexes and oligomers by nuclear magnetic resonance spectroscopy. NMR Biol Macromol B 339:238–258

    CAS  Google Scholar 

  5. Jung YS, Cai ML, Clore GM (2010) Solution structure of the IIA(chitobiose)-IIBchitobiose complex of the N,N′-diacetylchitobiose branch of the Escherichia coli phosphotransferase system. J Biol Chem 285:4173–4184

    Article  PubMed  CAS  Google Scholar 

  6. Finerty PJ, Mittermaier AK, Muhandiram R, Kay LE, Forman-Kay JD (2005) NMR dynamics-derived insights into the binding properties of a peptide interacting with an SH2 domain. Biochemistry 44:694–703

    Article  PubMed  CAS  Google Scholar 

  7. Boehr DD, McElheny D, Dyson HJ, Wright PE (2006) The dynamic energy landscape of dihydrofolate reductase catalysis. Science 313:1638–1642

    Article  PubMed  CAS  Google Scholar 

  8. Sugase K, Dyson HJ, Wright PE (2007) Mechanism of coupled folding and binding of an intrinsically disordered protein. Nature 447:1021–1025

    Article  PubMed  CAS  Google Scholar 

  9. Iwahara J, Clore GM (2006) Detecting transient intermediates in macromolecular binding by paramagnetic NMR. Nature 440:1227–1230

    Article  PubMed  CAS  Google Scholar 

  10. Meyer B, Peters T (2003) NMR spectroscopy techniques for screening and identifying ligand binding to protein receptors. Angew Chem Int Ed Engl 42:864–890

    Article  PubMed  CAS  Google Scholar 

  11. Shuker SB, Hajduk PJ, Meadows RP, Fesik SW (1996) Discovering high-affinity ligands for proteins: SAR by NMR. Science 274:1531–1534

    Article  PubMed  CAS  Google Scholar 

  12. Zarrine-Afsar A, Mittermaier A, Kay LE, Davidson AR (2006) Protein stabilization by specific binding of guanidinium to a functional arginine-binding surface on an SH3 domain. Protein Sci 15:162–170

    Article  PubMed  CAS  Google Scholar 

  13. Chen Y, Reizer J, Saier MH, Fairbrother WJ, Wright PE (1993) Mapping of the binding interfaces of the proteins of the bacterial phosphotransferase system, HPr and IIAglc. Biochemistry 32:32–37

    Article  PubMed  CAS  Google Scholar 

  14. Liu ZH et al (2000) Structural basis for binding of Smac/DIABLO to the XIAP BIR3 domain. Nature 408:1004–1008

    Article  PubMed  CAS  Google Scholar 

  15. Demers JP, Mittermaier A (2009) Binding mechanism of an SH3 domain studied by NMR and ITC. J Am Chem Soc 131:4355–4367

    Article  PubMed  CAS  Google Scholar 

  16. Schuck P (1996) Kinetics of ligand binding to receptor immobilized in a polymer matrix, as detected with an evanescent wave biosensor.1. A computer simulation of the influence of mass transport. Biophys J 70:1230–1249

    Article  PubMed  CAS  Google Scholar 

  17. Tochtrop GP, Richter K, Tang C, Toner JJ, Covey DF, Cistola DP (2002) Energetics by NMR: site-specific binding in a positively cooperative system. Proc Natl Acad Sci U S A 99:1847–1852

    Article  PubMed  CAS  Google Scholar 

  18. Freiburger LA, Baettig OM, Sprules T, Berghuis AM, Auclair K, Mittermaier AK (2011) Competing allosteric mechanisms modulate substrate binding in a dimeric enzyme. Nat Struct Mol Biol 18:288–294

    Article  PubMed  CAS  Google Scholar 

  19. Tugarinov V, Choy WY, Orekhov VY, Kay LE (2005) Solution NMR-derived global fold of a monomeric 82-kDa enzyme. Proc Natl Acad Sci U S A 102:622–627

    Article  PubMed  CAS  Google Scholar 

  20. Bodenhausen G, Ruben DJ (1980) Natural abundance nitrogen-15 NMR by enhanced heteronuclear spectroscopy. Chem Phys Lett 69:185–189

    Article  CAS  Google Scholar 

  21. 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 121:2331–2332

    Article  CAS  Google Scholar 

  22. Farrow NA, Zhang O, Forman-Kay JD, Kay LE (1994) A heteronuclear correlation experiment for simultaneous determination of 15 N longitudinal decay and chemical exchange rates of systems in slow equilibrium. J Biomol NMR 4:727–734

    Article  PubMed  CAS  Google Scholar 

  23. Gardner KH, Kay LE (1998) The use of 2H, 13C, 15 N multidimensional NMR to study the structure and dynamics of proteins. Annu Rev Biophys Biomol Struct 27:357–406

    Article  PubMed  CAS  Google Scholar 

  24. Marley J, Lu M, Bracken C (2001) A method for efficient isotopic labeling of recombinant proteins. J Biomol NMR 20:71–75

    Article  PubMed  CAS  Google Scholar 

  25. Bai YW, Milne JS, Mayne L, Englander SW (1993) Primary structure effects on peptide group hydrogen exchange. Protein Struct Funct Genet 17:75–86

    Article  CAS  Google Scholar 

  26. Sugase K, Lansing JC, Dyson HJ, Wright PE (2007) Tailoring relaxation dispersion experiments for fast-associating protein complexes. J Am Chem Soc 129:13406–13407

    Article  PubMed  CAS  Google Scholar 

  27. Johnson BA, Blevins RA (1994) NMR view – a computer program for the visualization and analysis of NMR data. J Biomol NMR 4:603–614

    Article  PubMed  CAS  Google Scholar 

  28. van Beek JD (2007) matNMR: a flexible toolbox for processing, analyzing and visualizing magnetic resonance data in Matlab. J Magn Reson 187:19–26

    Article  PubMed  Google Scholar 

  29. Vranken WF, Boucher W, Stevens TJ, Fogh RH, Pajon A, Llinas P, Ulrich EL, Markley JL, Ionides J, Laue ED (2005) The CCPN data model for NMR spectroscopy: development of a software pipeline. Protein Struct Funct Bioinf 59:687–696

    Article  CAS  Google Scholar 

  30. Gunther UL, Ludwig C, Ruterjans H (2000) NMRLAB – advanced NMR data processing in MATLAB. J Magn Reson 145:201–208

    Article  PubMed  CAS  Google Scholar 

  31. 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

    Article  PubMed  CAS  Google Scholar 

  32. Gunther UL, Schaffhausen B (2002) NMRKIN: simulating line shapes from two-dimensional spectra of proteins upon ligand binding. J Biomol NMR 22:201–209

    Article  PubMed  CAS  Google Scholar 

  33. Vetterling WT, Press WH, Teukolsky SA, Flannery BR (1988) Numerical recipes in C. Cambridge University Press, Cambridge

    Google Scholar 

  34. Langmuir I (1916) The constitution and fundamental properties of solids and liquids part I solids. J Am Chem Soc 38:2221–2295

    Article  CAS  Google Scholar 

  35. Wiseman T, Williston S, Brandts JF, Lin LN (1989) Rapid measurement of binding constants and heats of binding using a new titration calorimeter. Anal Biochem 179:131–137

    Article  PubMed  CAS  Google Scholar 

  36. Bain AD (2003) Chemical exchange in NMR. Prog NMR Spect 43:63–103

    Article  CAS  Google Scholar 

  37. Gutowsky HS, Saika A (1953) Dissociation, chemical exchange, and the proton magnetic resonance in some aqueous electrolytes. J Chem Phys 21:1688–1694

    Article  CAS  Google Scholar 

  38. McConnell HM (1958) Reaction rates by nuclear magnetic resonance. J Chem Phys 28:430–431

    Article  CAS  Google Scholar 

  39. Rogers MT, Woodbrey JC (1962) Proton magnetic resonance study of hindered internal rotation in some substituted N, N-dimethylamides. J Phys Chem 66:540–562

    Article  CAS  Google Scholar 

  40. Gupta RK, Pitner TP, Wasylishen R (1974) Fourier transform NMR of exchanging chemical systems. J Magn Reson 13:383–385

    CAS  Google Scholar 

  41. Mittag T, Schaffhausen B, Gunther UL (2004) Tracing kinetic intermediates during ligand binding. J Am Chem Soc 126:9017–9023

    Article  PubMed  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  44. Palmer AG, Kroenke CD, Loria JP (2001) Nuclear magnetic resonance methods for quantifying microsecond-to-millisecond motions in biological macromolecules. NMR Biol Macromol B 339:204–238

    CAS  Google Scholar 

  45. 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 41:442–451

    Article  PubMed  CAS  Google Scholar 

  46. Hahn EL (1950) Spin echoes. Phys Rev 80:580–594

    Article  Google Scholar 

  47. Esadze A, Li DW, Wang TZ, Bruschweiler R, Iwahara J (2011) Dynamics of lysine side-chain amino groups in a protein studied by heteronuclear H-1-N-15 NMR spectroscopy. J Am Chem Soc 133:909–919

    Article  PubMed  CAS  Google Scholar 

  48. Ishima R, Baber J, Louis JM, Torchia DA (2004) Carbonyl carbon transverse relaxation dispersion measurements and ms-mu s timescale motion in a protein hydrogen bond network. J Biomol NMR 29:187–198

    Article  PubMed  CAS  Google Scholar 

  49. 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  PubMed  CAS  Google Scholar 

  50. Korzhnev DM, Neudecker P, Mittermaier A, Orekhov VY, Kay LE (2005) Multiple-site exchange in proteins studied with a suite of six NMR relaxation dispersion experiments: an application to the folding of a Fyn SH3 domain mutant. J Am Chem Soc 127:15602–15611

    Article  PubMed  CAS  Google Scholar 

  51. Mulder FAA, Skrynnikov NR, Hon B, Dahlquist FW, Kay LE (2001) Measurement of slow (mu s-ms) time scale dynamics in protein side chains by N-15 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  PubMed  CAS  Google Scholar 

  52. Skrynnikov NR, Mulder FAA, Hon B, Dahlquist FW, Kay LE (2001) Probing slow time scale dynamics at methyl-containing side chains in proteins by relaxation dispersion NMR measurements: application to methionine residues in a cavity mutant of T4 lysozyme. J Am Chem Soc 123:4556–4566

    Article  PubMed  CAS  Google Scholar 

  53. Lundstrom P, Lin H, Kay LE (2009) Measuring C-13(beta) chemical shifts of invisible excited states in proteins by relaxation dispersion NMR spectroscopy. J Biomol NMR 44:139–155

    Article  PubMed  Google Scholar 

  54. Baldwin AJ, Hansen DF, Vallurupalli P, Kay LE (2009) Measurement of methyl axis orientations in invisible, excited states of proteins by relaxation dispersion NMR spectroscopy. J Am Chem Soc 131:11939–11948

    Article  PubMed  CAS  Google Scholar 

  55. Hansen DF, Vallurupalli P, Kay LE (2009) Measurement of methyl group motional parameters of invisible, excited protein states by NMR spectroscopy. J Am Chem Soc 131:12745–12754

    Article  PubMed  CAS  Google Scholar 

  56. 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 U S A 104:18473–18477

    Article  PubMed  CAS  Google Scholar 

  57. Palmer AG, Massi F (2006) Characterization of the dynamics of biomacromolecules using rotating-frame spin relaxation NMR spectroscopy. Chem Rev 106:1700–1719

    Article  PubMed  CAS  Google Scholar 

  58. Hansen DF, Vallurupalli P, Kay LE (2008) An improved N-15 relaxation dispersion experiment for the measurement of millisecond time-scale dynamics in proteins. J Phys Chem B 112:5898–5904

    Article  PubMed  CAS  Google Scholar 

  59. Ishima R, Torchia DA (2006) Accuracy of optimized chemical-exchange parameters derived by fitting CPMG R2 dispersion profiles when R2(0a) = R2(0b). J Biomol NMR 34:209–219

    Article  PubMed  CAS  Google Scholar 

  60. 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  PubMed  CAS  Google Scholar 

  61. Carver JP, Richards RE (1972) General 2-site solution for chemical exchange produced dependence of T2 upon Carr-Purcell pulse separation. J Magn Reson 6:89–105

    CAS  Google Scholar 

  62. Davis DG, Perlman ME, London RE (1994) Direct measurements of the dissociation rate constant for inhibitor enzyme complexes via the T1rho and T2(CPMG) methods. J Magn Reson B 104:266–275

    Article  PubMed  CAS  Google Scholar 

  63. Luz Z, Meiboom S (1963) Nuclear magnetic resonance study of protolysis of trimethylammonium ion in aqueous solution—order of reaction with respect to solvent. J Chem Phys 39:366–371

    Article  CAS  Google Scholar 

  64. Kovrigin EL, Kempf JG, Grey MJ, Loria JP (2006) Faithful estimation of dynamics parameters from CPMG relaxation dispersion measurements. J Magn Reson 180:93–104

    Article  PubMed  CAS  Google Scholar 

  65. Millet O, Loria JP, Kroenke CD, Pons M, Palmer AG (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  CAS  Google Scholar 

  66. Mulder FAA, Mittermaier A, Hon B, Dahlquist FW, Kay LE (2001) Studying excited states of proteins by NMR spectroscopy. Nat Struct Biol 8:932–935

    Article  PubMed  CAS  Google Scholar 

  67. Findeisen M, Brand T, Berger S (2007) A H-1-NMR thermometer suitable for cryoprobes. Magn Reson Chem 45:175–178

    Article  PubMed  CAS  Google Scholar 

  68. Hindman JC (1966) Proton resonance shift of water in gas and liquid states. J Chem Phys 44:4582–4593

    Article  CAS  Google Scholar 

  69. Renzoni DA, Pugh DJR, Siligardi G, Das P, Morton CJ, Rossi C, Waterfield MD, Campbell ID, Ladbury JE (1996) Structural and thermodynamic characterization of the interaction of the SH3 domain from Fyn with the proline-rich binding site on the p85 subunit of PI3-kinase. Biochemistry 35:15646–15653

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Chemistry, McGill University, Montreal, QC, Canada

    Anthony Mittermaier & Erick Meneses

Authors
  1. Anthony Mittermaier
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Erick Meneses
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Birbeck, University of London, ISMB Biophysics Centre, Institute of Str, Malet Street, London, WC1E 7HX, United Kingdom

    Mark A. Williams

  2. Birkbeck, University of London, School of Crystallography, ISMB Biophysics Centre, Institute of Str, Malet Street, London, WC1E 7HX, United Kingdom

    Tina Daviter

Rights and permissions

Reprints and permissions

Copyright information

© 2013 Springer Science+Business Media New York

About this protocol

Cite this protocol

Mittermaier, A., Meneses, E. (2013). Analyzing Protein–Ligand Interactions by Dynamic NMR Spectroscopy. In: Williams, M., Daviter, T. (eds) Protein-Ligand Interactions. Methods in Molecular Biology, vol 1008. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-398-5_9

Download citation

  • DOI: https://doi.org/10.1007/978-1-62703-398-5_9

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-397-8

  • Online ISBN: 978-1-62703-398-5

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation