AMPK Methods and Protocols

Chapter 6

Applications of NMR and ITC for the Study of the Kinetics

of Carbohydrate Binding by AMPKβ-Subunit

Carbohydrate-Binding Modules

Paul R. Gooley, Ann Koay, and Jesse I. Mobbs

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 theβ1- and
β2-carbohydrate-binding modules of AMP-activated protein kinase.

Key wordsCarbohydrate, CPMG relaxation dispersion, Isothermal titration calorimetry, Ligand

binding, Nuclear magnetic resonance, ZZ-exchange

1 Introduction

AMPK, composed of differentβ-isoform subunits, binds carbohy-

drates with varying affinities due to the carbohydrate-binding mod-

ules (β1- orβ2-CBM) that they carry [1–3]. Theβ2-CBM binds

carbohydrate with greater affinity than those with theβ1-CBM. To

gain a better understanding of this difference, the thermodynamics

and kinetics of binding can be characterized by a variety of analytical

methods. Initial characterization of the affinities by fluorescence

spectrophotometry and NMR spectroscopy showed dissociation

constants (Kd) of 0.5–10μM for various carbohydrates [2]. Further

tests suggested the molecular mass of the CBMs (~10 kDa), small

ligand size (~1 kDa), and these affinities were not suitable for using

surface plasmon resonance (SPR), an excellent method for obtain-

ing kinetics and thermodynamic data [4]. On the other hand, as

Dietbert Neumann and Benoit Viollet (eds.),AMPK:MethodsandProtocols, Methods in Molecular Biology, vol. 1732,
https://doi.org/10.1007/978-1-4939-7598-3_6,©Springer Science+Business Media, LLC 2018

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