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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
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1732)

Abstract

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 

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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

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