AMPK pp 87-98 | Cite as

Applications of NMR and ITC for the Study of the Kinetics of Carbohydrate Binding by AMPK β-Subunit Carbohydrate-Binding Modules

  • Paul R. GooleyEmail author
  • Ann Koay
  • Jesse I. Mobbs
Part of the Methods in Molecular Biology book series (MIMB, volume 1732)


Understanding the kinetics of proteins interacting with their ligands is important for characterizing molecular mechanism. However, it can be difficult to determine the extent and nature of these interactions for weakly formed protein-ligand complexes that have lifetimes of micro- to milliseconds. Nuclear magnetic resonance (NMR) spectroscopy is a powerful solution-based method for the atomic-level analysis of molecular interactions on a wide range of timescales, including micro- to milliseconds. Recently the combination of thermodynamic experiments using isothermal titration calorimetry (ITC) with kinetic measurements using ZZ-exchange and CPMG relaxation dispersion NMR spectroscopy have been used to determine the kinetics of weakly interacting protein systems. This chapter describes the application of ITC and NMR to understand the differences in the kinetics of carbohydrate binding by the β1- and β2-carbohydrate-binding modules of AMP-activated protein kinase.

Key words

Carbohydrate CPMG relaxation dispersion Isothermal titration calorimetry Ligand binding Nuclear magnetic resonance ZZ-exchange 


  1. 1.
    Koay A, Rimmer KA, Mertens HD et al (2007) Oligosaccharide recognition and binding to the carbohydrate binding module of AMP-activated protein kinase. FEBS Lett 581:5055–5059CrossRefPubMedGoogle Scholar
  2. 2.
    Koay A, Woodcroft B, Petrie EJ et al (2010) AMPK beta subunits display isoform specific affinities for carbohydrates. FEBS Lett 584:3499–3503CrossRefPubMedGoogle Scholar
  3. 3.
    Mobbs JI, Koay A, Di Paolo A et al (2015) Determinants of oligosaccharide specificity of the carbohydrate-binding modules of AMP-activated protein kinase. Biochem J 468:245–257CrossRefPubMedGoogle Scholar
  4. 4.
    Navratilova I, Papalia GA, Rich RL et al (2007) Thermodynamic benchmark study using Biacore technology. Anal Biochem 364:67–77CrossRefPubMedGoogle Scholar
  5. 5.
    Demers JP, Mittermaier A (2009) Binding mechanism of an SH3 domain studied by NMR and ITC. J Am Chem Soc 131:4355–4367CrossRefPubMedGoogle Scholar
  6. 6.
    Velazquez-Campoy A, Ohtaka H, Nezami A et al. (2004) Isothermal titration calorimetry. Curr Protoc Cell Biol. Chapter 17:Unit 17 18Google Scholar
  7. 7.
    Kleckner IR, Foster MP (2011) An introduction to NMR-based approaches for measuring protein dynamics. Biochim Biophys Acta 1814:942–968CrossRefPubMedGoogle Scholar
  8. 8.
    Henzler-Wildman K, Kern D (2007) Dynamic personalities of proteins. Nature 450:964–972CrossRefPubMedGoogle Scholar
  9. 9.
    Vafabakhsh R, Levitz J, Isacoff EY (2015) Conformational dynamics of a class C G-protein-coupled receptor. Nature 524:497–501CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Farrow NA, Zhang O, Forman-Kay JD et al (1994) A heteronuclear correlation experiment for simultaneous determination of 15N longitudinal decay and chemical exchange rates of systems in slow equilibrium. J Biomol NMR 4:727–734CrossRefPubMedGoogle Scholar
  11. 11.
    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
  12. 12.
    Korzhnev DM, Salvatella X, Vendruscolo M et al (2004) Low-populated folding intermediates of Fyn SH3 characterized by relaxation dispersion NMR. Nature 430:586–590CrossRefPubMedGoogle Scholar
  13. 13.
    Meneses E, Mittermaier A (2014) Electrostatic interactions in the binding pathway of a transient protein complex studied by NMR and isothermal titration calorimetry. J Biol Chem 289:27911–27923CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Bieri M, Mobbs JI, Koay A et al (2012) AMP-activated protein kinase beta-subunit requires internal motion for optimal carbohydrate binding. Biophys J 102:305–314CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Gill SC, Von Hippel PH (1989) Calculation of protein extinction coefficients from amino acid sequence data. Anal Biochem 182:319–326CrossRefPubMedGoogle Scholar
  16. 16.
    Delaglio F, Grzesiek S, Vuister GW et al (1995) NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR 6:277–293CrossRefPubMedGoogle Scholar
  17. 17.
    Sugase K, Lansing JC, Dyson HJ et al (2007) Tailoring relaxation dispersion experiments for fast-associating protein complexes. J Am Chem Soc 129:13406–13407CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Cai M, Huang Y, Sakaguchi K et al (1998) An efficient and cost-effective isotope labeling protocol for proteins expressed in Escherichia Coli. J Biomol NMR 11:97–102CrossRefPubMedGoogle Scholar
  19. 19.
    Marley J, Lu M, Bracken C (2001) A method for efficient isotopic labeling of recombinant proteins. J Biomol NMR 20:71–75CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  1. 1.Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology InstituteThe University of MelbourneParkvilleAustralia
  2. 2.Experimental Therapeutics CentreAgency for Science Technology and ResearchSingaporeSingapore
  3. 3.Department of Biochemistry and Molecular Biology, School of Biomedical SciencesMonash UniversityClaytonAustralia

Personalised recommendations