Mammalian membrane proteins are invariably more unstable than bacterial membrane proteins after detergent solubilisation and purification, which makes them far more difficult to crystallise. We have developed a generic methodology based upon alanine scanning mutagenesis and a selection strategy to improve the thermostability of any membrane protein. This was used to stabilise the b1 adrenergic receptor. The thermostabilised mutant bAR-m23 contained 6 mutations and was more stable than the wild type protein by 21˚C. This allowed the purification and crystallisation of the protein in short-chain detegents, which normally causes the receptor to aggregate. The structure clearly shows the ligand cyanopindolol in the receptor binding pocket and will be compared with the recently determined structures of the b2 adrenergic receptor crystallised in the presence of carazolol.
Maria J. Serrano-Vega, Francesca Magnani, Yoko Shibata & Christopher G. Tate* (2008) Conformational thermostabilisation of the ß-adrenergic receptor in a detergent-resistant form. Proc Natl Acad Sci USA 105, 877-882
Tony Warne, Maria J. Serrano-Vega, Jillian G. Baker#, Rouslan Moukhametzianov, Patricia C. Edwards, Richard Henderson, Andrew G.W. Leslie, Christopher G. Tate* & Gebhard F.X. Schertler* (2008) Structure of ß 1 adrenergic G protein-coupled receptor. Submitted.
# School of Medicine, Institute of Cell Signalling, Nottingham NG7 2UH, UK