Nuts and Bolts of CF3 and CH3 NMR Toward the Understanding of Conformational Exchange of GPCRs

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1335)

Abstract

With the advent of efficient protein expression and functional purification protocols, it is now possible to reconstitute many G protein-coupled receptors (GPCRs) in detergent micelles at concentrations of 25 μM or more. Such concentrations are sufficient for studies of conformational states and dynamics relating to function and the mechanism of activation of GPCRs, using solution state NMR. In particular, methyl spectroscopy, in the form of one-dimensional 19F NMR or two-dimensional (1H,13C) NMR, provides high fidelity spectra which reveal detailed features associated with conformational states and their lifetimes, as a function of ligand. While X-ray crystallography provides exquisitely detailed structures of lowest energy states associated with ligands, G proteins, and other proteins, NMR is able to validate such states, while providing insight into higher energy states that form part of the conformational landscape and are involved in activation. Through relaxation experiments spanning microseconds to seconds, lifetimes of these functional states can often be measured. By determining the effect of ligands on both equilibrium populations and rates of interconversion between states, it becomes possible to understand activation in terms of an ensemble description and in turn relate the ensemble to pharmaceutical phenomena.

Key words

GPCR 19F NMR Methyl spectroscopy Isotope labeling Protein dynamics 
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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Department of ChemistryUniversity of Toronto (UTM)MississaugaCanada
  2. 2.Department of BiochemistryUniversity of TorontoTorontoCanada

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