A mutagenesis and screening strategy to generate optimally thermostabilized membrane proteins for structural studies.

Autor: Magnani F; MRC Laboratory of Molecular Biology, Cambridge, UK., Serrano-Vega MJ; MRC Laboratory of Molecular Biology, Cambridge, UK., Shibata Y; MRC Laboratory of Molecular Biology, Cambridge, UK., Abdul-Hussein S; MRC Laboratory of Molecular Biology, Cambridge, UK., Lebon G; MRC Laboratory of Molecular Biology, Cambridge, UK., Miller-Gallacher J; MRC Laboratory of Molecular Biology, Cambridge, UK., Singhal A; MRC Laboratory of Molecular Biology, Cambridge, UK., Strege A; MRC Laboratory of Molecular Biology, Cambridge, UK., Thomas JA; MRC Laboratory of Molecular Biology, Cambridge, UK., Tate CG; MRC Laboratory of Molecular Biology, Cambridge, UK.
Jazyk: angličtina
Zdroj: Nature protocols [Nat Protoc] 2016 Aug; Vol. 11 (8), pp. 1554-71. Date of Electronic Publication: 2016 Jul 28.
DOI: 10.1038/nprot.2016.088
Abstrakt: 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.
Competing Interests: The authors declare competing financial interests.
Databáze: MEDLINE
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