Development and crystallization of a minimal thermostabilised G protein-coupled receptor.
Structure determination of G protein-coupled receptors is still in its infancy and many factors affect whether crystals are obtained and whether the diffraction is of sufficient quality for structure determination. We recently solved the structure of a thermostabilised turkey beta 1-adrenergic receptor by crystallization in the presence of the detergent octylthioglucoside. Three factors were essential for this success. Firstly, truncations were required at the N-terminus to give optimal expression. Secondly, 6 thermostabilising point mutations were incorporated to make the receptor sufficiently stable in short-chain detergents to allow crystallization. Thirdly, truncations at the C-terminus and within cytoplasmic loop 3, in combination with the removal of the palmitoylation site, were required to obtain well-diffracting crystals in octylthioglucoside. Here, we describe the strategy employed and the utility of thermostability assays in assessing how point mutations, truncations, detergents and ligands combine to develop a construct that forms diffraction-grade crystals.
Warne
Tony
T
MRC Laboratory of Molecular Biology, Structural Studies, Hills Road, Cambridge CB2 0QH, UK.
Serrano-Vega
Maria J
MJ
Tate
Christopher G
CG
Schertler
Gebhard F X
GF
D000595:Amino Acid Sequence; D000818:Animals; D002846:Chromatography, Affinity; D003460:Crystallization; D008969:Molecular Sequence Data; D016296:Mutagenesis; D055550:Protein Stability; D018342:Receptors, Adrenergic, beta-1 / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D012995:Solubility; D013696:Temperature; D014422:Turkeys
D018342:Receptors, Adrenergic, beta-1