Membrane Protein Structure Determination pp 141-168

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

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

  • Vadim Cherezov
  • Enrique Abola
  • Raymond C. Stevens
Protocol

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.

Keywords

GPCR Lipidic cubic phase Membrane proteins Fluorescence recovery after photobleaching Crystallization Crystallography Minibeam 

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Vadim Cherezov
    • 1
  • Enrique Abola
    • 1
  • Raymond C. Stevens
    • 1
  1. 1.Department of Molecular BiologyThe Scripps Research InstituteLa JollaUSA

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