title: A mutagenesis and screening strategy to generate optimally thermostabilized membrane proteins for structural studies.
abstract: The thermostability of an integral membrane protein (MP) in detergent solution is a key parameter that dictates the likelihood of obtaining well-diffracting crystals that are suitable for structure determination. However, many mammalian MPs are too unstable for crystallization. We developed a thermostabilization strategy based on systematic mutagenesis coupled to a radioligand-binding thermostability assay that can be applied to receptors, ion channels and transporters. It takes ∼6-12 months to thermostabilize a G-protein-coupled receptor (GPCR) containing 300 amino acid (aa) residues. The resulting thermostabilized MPs are more easily crystallized and result in high-quality structures. This methodology has facilitated structure-based drug design applied to GPCRs because it is possible to determine multiple structures of the thermostabilized receptors bound to low-affinity ligands. Protocols and advice are given on how to develop thermostability assays for MPs and how to combine mutations to make an optimally stable mutant suitable for structural studies. The steps in the procedure include the generation of ∼300 site-directed mutants by Ala/Leu scanning mutagenesis, the expression of each mutant in mammalian cells by transient transfection and the identification of thermostable mutants using a thermostability assay that is based on binding of an (125)I-labeled radioligand to the unpurified, detergent-solubilized MP. Individual thermostabilizing point mutations are then combined to make an optimally stable MP that is suitable for structural biology and other biophysical studies.
authors.affiliation: MRC Laboratory of Molecular Biology, Cambridge, UK.
authors.forename: Francesca
authors.initials: F
authors.lastname: Magnani
authors.affiliation: MRC Laboratory of Molecular Biology, Cambridge, UK.
authors.forename: Maria J
authors.initials: MJ
authors.lastname: Serrano-Vega
authors.affiliation: MRC Laboratory of Molecular Biology, Cambridge, UK.
authors.forename: Yoko
authors.initials: Y
authors.lastname: Shibata
authors.affiliation: MRC Laboratory of Molecular Biology, Cambridge, UK.
authors.forename: Saba
authors.initials: S
authors.lastname: Abdul-Hussein
authors.affiliation: MRC Laboratory of Molecular Biology, Cambridge, UK.
authors.forename: Guillaume
authors.initials: G
authors.lastname: Lebon
authors.affiliation: MRC Laboratory of Molecular Biology, Cambridge, UK.
authors.forename: Jennifer
authors.identifier: 0000-0003-0539-0223
authors.initials: J
authors.lastname: Miller-Gallacher
authors.affiliation: MRC Laboratory of Molecular Biology, Cambridge, UK.
authors.forename: Ankita
authors.initials: A
authors.lastname: Singhal
authors.affiliation: MRC Laboratory of Molecular Biology, Cambridge, UK.
authors.forename: Annette
authors.initials: A
authors.lastname: Strege
authors.affiliation: MRC Laboratory of Molecular Biology, Cambridge, UK.
authors.forename: Jennifer A
authors.identifier: 0000-0003-1065-4131
authors.initials: JA
authors.lastname: Thomas
authors.affiliation: MRC Laboratory of Molecular Biology, Cambridge, UK.
authors.forename: Christopher G
authors.identifier: 0000-0002-2008-9183
authors.initials: CG
authors.lastname: Tate
mesh_terms: D000595:Amino Acid Sequence; D003902:Detergents / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics*; D008958:Models, Molecular; D016296:Mutagenesis; D009154:Mutation; D011487:Protein Conformation; D055550:Protein Stability; D012995:Solubility; D013696:Temperature
chemical_list: D003902:Detergents; D008565:Membrane Proteins