Crystallization of G protein-coupled receptors

Methods Cell Biol. 2013;117:451-68. doi: 10.1016/B978-0-12-408143-7.00024-4.

Abstract

Oligomerization is one of several mechanisms that can regulate the activity of G protein-coupled receptors (GPCRs), but little is known about the structure of GPCR oligomers. Crystallography and NMR are the only methods able to reveal the details of receptor-receptor interactions at an atomic level, and several GPCR homodimers already have been described from crystal structures. Two clusters of symmetric interfaces have been identified from these structures that concur with biochemical data, one involving helices I, II, and VIII and the other formed mainly by helices V and VI. In this chapter, we describe the protocols used in our laboratory for the crystallization of rhodopsin and the β2-adrenergic receptor (β2-AR). For bovine rhodopsin, we developed a new purification strategy including a (NH4)2SO4-induced phase separation that proved essential to obtain crystals of photoactivated rhodopsin containing parallel dimers. Crystallization of native bovine rhodopsin was achieved by the classic vapor-diffusion technique. For β2-AR, we developed a purification strategy based on previously published protocols employing a lipidic cubic phase to obtain diffracting crystals of a β2-AR/T4-lysozyme chimera bound to the antagonist carazolol.

Keywords: Ammonium sulfate; Crystallization; G protein-coupled receptor; Lipidic cubic phase; Purification; Rhodopsin; β2-Adrenergic receptor.

MeSH terms

  • Ammonium Sulfate / chemistry
  • Animals
  • Cattle
  • Centrifugation, Density Gradient
  • Chromatography, Affinity
  • Chromatography, Gel
  • Crystallization
  • Crystallography, X-Ray
  • Glucosides / chemistry
  • Muramidase / chemistry
  • Muramidase / genetics
  • Propanolamines / chemistry
  • Protein Multimerization
  • Protein Structure, Secondary
  • Receptors, Adrenergic, beta-2 / chemistry*
  • Receptors, Adrenergic, beta-2 / genetics
  • Receptors, Adrenergic, beta-2 / isolation & purification
  • Recombinant Fusion Proteins / chemistry*
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / isolation & purification
  • Rhodopsin / chemistry*
  • Rhodopsin / isolation & purification
  • Rod Cell Outer Segment / chemistry*
  • Sf9 Cells
  • Spodoptera
  • Viral Proteins / chemistry
  • Viral Proteins / genetics
  • Zinc Acetate / chemistry

Substances

  • Glucosides
  • Propanolamines
  • Receptors, Adrenergic, beta-2
  • Recombinant Fusion Proteins
  • Viral Proteins
  • carazolol
  • Rhodopsin
  • Muramidase
  • Zinc Acetate
  • Ammonium Sulfate