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

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G Protein-Coupled Receptors in Drug Discovery

Part of the book series: Methods in Molecular Biology ((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.

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Authors and Affiliations

  1. Department of Chemistry, University of Toronto (UTM), 3359 Mississauga Road North, Mississauga, ON, Canada, L5L 1C6

    R. Scott Prosser & Tae Hun Kim

  2. Department of Biochemistry, University of Toronto, 1 King’s College Circle, Toronto, ON, Canada, M5S 1A8

    R. Scott Prosser

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  1. R. Scott Prosser
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  2. Tae Hun Kim
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Correspondence to R. Scott Prosser .

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Editors and Affiliations

  1. Department of Structural and Chemical Biology, Icahn School of Medicine at Mount Sinai, New York, New York, USA

    Marta Filizola

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Prosser, R.S., Kim, T.H. (2015). Nuts and Bolts of CF3 and CH3 NMR Toward the Understanding of Conformational Exchange of GPCRs. In: Filizola, M. (eds) G Protein-Coupled Receptors in Drug Discovery. Methods in Molecular Biology, vol 1335. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2914-6_4

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  • DOI: https://doi.org/10.1007/978-1-4939-2914-6_4

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2913-9

  • Online ISBN: 978-1-4939-2914-6

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