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Recent Progress in the Structure Determination of GPCRs, a Membrane Protein Family with High Potential as Pharmaceutical Targets

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Membrane Protein Structure Determination

Part of the book series: Methods in Molecular Biology ((MIMB,volume 654))

Abstract

G protein-coupled receptors (GPCRs) constitute a highly diverse and ubiquitous family of integral membrane proteins, transmitting signals inside the cells in response to an assortment of disparate extracellular stimuli. Their strategic location on the cell surface and their involvement in crucial cellular and physiological processes turn these receptors into highly important pharmaceutical targets. Recent technological developments aimed at stabilization and crystallization of these receptors have led to significant breakthroughs in GPCR structure determination efforts. One of the successful approaches involved receptor stabilization with the help of a fusion partner combined with crystallization in lipidic cubic phase (LCP). The success of using an LCP matrix for crystallization is generally attributed to the creation of a more native, membrane-like stabilizing environment for GPCRs just prior to nucleation and to the formation of type I crystal lattices, thus generating highly ordered and strongly diffracting crystals. Here we describe protocols for reconstituting purified GPCRs in LCP, performing pre-crystallization assays, setting up crystallization trials in manual mode, detecting crystallization hits, optimizing crystallization conditions, harvesting, and collecting crystallographic data The protocols provide a sensible framework for approaching crystallization of stabilized GPCRs in LCP, however, as in any crystallization experiment, extensive screening and optimization of crystallization conditions as well as optimization of protein construct and purification steps are required. The process remains risky and these protocols do not necessarily guarantee success.

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Acknowledgements

This work was supported in part by the NIH Roadmap Initiative grant P50 GM073197 (JCIMPT) and the Protein Structure Initiative grant U54 GM074961 (ATCG3D). The authors acknowledge contributions from colleagues Michael A. Hanson, Wei Liu, Jeffrey Liu, Mark Griffith, Ellen Chien, Veli-Pekka Jaakola, Chris Roth and Peter Kuhn.

The authors acknowledge the support of Janet Smith, Robert Fischetti, and the GM/CA-CAT team at the Advanced Photon Source, for assistance in development and use of the minibeam and beamtime. The GM/CA-CAT beamline (23-ID) is supported by the National Cancer Institute (Y1-CO-1020) and the National Institute of General Medical Sciences (Y1-GM-1104).

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Authors and Affiliations

  1. Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA, USA

    Vadim Cherezov, Enrique Abola & Raymond C. Stevens

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  1. Vadim Cherezov
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  2. Enrique Abola
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  3. Raymond C. Stevens
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Editors and Affiliations

  1. , Université Paris Diderot - Paris 7, INSERM U773/CRB3, rue Henri Huchard 16, Paris, 75018, France

    Jean-Jacques Lacapère

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Cherezov, V., Abola, E., Stevens, R.C. (2010). Recent Progress in the Structure Determination of GPCRs, a Membrane Protein Family with High Potential as Pharmaceutical Targets. In: Lacapère, JJ. (eds) Membrane Protein Structure Determination. Methods in Molecular Biology, vol 654. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-762-4_8

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  • DOI: https://doi.org/10.1007/978-1-60761-762-4_8

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-60761-761-7

  • Online ISBN: 978-1-60761-762-4

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