title: Engineering an ultra-thermostable β(1)-adrenoceptor.
abstract: Conformational thermostabilisation of G-protein-coupled receptors is a successful strategy for their structure determination. The thermostable mutants tolerate short-chain detergents, such as octylglucoside and nonylglucoside, which are ideal for crystallography, and in addition, the receptors are preferentially in a single conformational state. The first thermostabilised receptor to have its structure determined was the β(1)-adrenoceptor mutant β(1)AR-m23 bound to the antagonist cyanopindolol, and recently, additional structures have been determined with agonist bound. Here, we describe further stabilisation of β(1)AR-m23 by the addition of three thermostabilising mutations (I129V, D322K, and Y343L) to make a mutant receptor that is 31 °C more thermostable than the wild-type receptor in dodecylmaltoside and is 13 °C more thermostable than β(1)AR-m23 in nonylglucoside. Although a number of thermostabilisation methods were tried, including rational design of disulfide bonds and engineered zinc bridges, the two most successful strategies to improve the thermostability of β(1)AR-m23 were an engineered salt bridge and leucine scanning mutagenesis. The three additional thermostabilising mutations did not significantly affect the pharmacological properties of β(1)AR-m23, but the new mutant receptor was significantly more stable in short-chain detergents such as heptylthioglucoside and denaturing detergents such as SDS.
authors: MRC Laboratory of Molecular Biology, Cambridge CB2 0QH, UK.
authors: Jennifer L
authors: JL
authors: Miller
authors: Christopher G
authors: CG
authors: Tate
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D018360:Crystallography, X-Ray; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D010869:Pindolol / Q000031:analogs & derivatives / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D015202:Protein Engineering; D055550:Protein Stability; D011869:Radioligand Assay; D018342:Receptors, Adrenergic, beta-1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled; D013696:Temperature; D014421:Turkey; D015032:Zinc / Q000378:metabolism
chemical_list: D004220:Disulfides; D018342:Receptors, Adrenergic, beta-1; D043562:Receptors, G-Protein-Coupled; C047975:cyanopindolol; D010869:Pindolol; D015032:Zinc