N-terminal T4 lysozyme fusion facilitates crystallization of a G protein coupled receptor

PLoS One. 2012;7(10):e46039. doi: 10.1371/journal.pone.0046039. Epub 2012 Oct 4.

Abstract

A highly crystallizable T4 lysozyme (T4L) was fused to the N-terminus of the β(2) adrenergic receptor (β(2)AR), a G-protein coupled receptor (GPCR) for catecholamines. We demonstrate that the N-terminal fused T4L is sufficiently rigid relative to the receptor to facilitate crystallogenesis without thermostabilizing mutations or the use of a stabilizing antibody, G protein, or protein fused to the 3rd intracellular loop. This approach adds to the protein engineering strategies that enable crystallographic studies of GPCRs alone or in complex with a signaling partner.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Bacteriophage T4 / enzymology*
  • Binding Sites
  • Binding, Competitive
  • Crystallization
  • Crystallography, X-Ray
  • Dihydroalprenolol / chemistry
  • Dihydroalprenolol / metabolism
  • Humans
  • Models, Molecular
  • Molecular Sequence Data
  • Muramidase / chemistry*
  • Muramidase / genetics
  • Muramidase / metabolism
  • Mutation
  • Protein Binding
  • Protein Conformation
  • Protein Engineering / methods
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Receptors, Adrenergic, beta-2 / chemistry*
  • Receptors, Adrenergic, beta-2 / genetics
  • Receptors, Adrenergic, beta-2 / metabolism
  • Receptors, G-Protein-Coupled / chemistry*
  • Receptors, G-Protein-Coupled / genetics
  • Receptors, G-Protein-Coupled / metabolism
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Sf9 Cells
  • Tritium
  • Viral Proteins / chemistry*
  • Viral Proteins / genetics
  • Viral Proteins / metabolism

Substances

  • Receptors, Adrenergic, beta-2
  • Receptors, G-Protein-Coupled
  • Recombinant Fusion Proteins
  • Viral Proteins
  • Tritium
  • Dihydroalprenolol
  • Muramidase