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

Nature Protocols volume 11, pages 1554–1571 (2016)Cite this article

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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 125I-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.

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Figure 1: Flowchart illustrating the thermostabilization strategy.
Figure 2: Optimization of transient transfection in HEK293 cells.
Figure 3: Different formats of the thermostability assays.
Figure 4: Development of a thermostability assay for the serotonin transporter.
Figure 5: Thermostabilization of the β1-adrenergic receptor.
Figure 6: Thermostabilization of the adenosine A2A receptor in the agonist-bound conformation.
Figure 7: Thermostability of the ultrastable β1AR mutant, JM50.

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Acknowledgements

Funding for the thermostabilization of membrane proteins in the laboratory of C.G.T. was from the Medical Research Council (MRC U105197215), Medical Research Council Technology Development Gap Fund, Pfizer, Heptares Therapeutics and an ERC Advanced Grant (EMPSI 339995). We thank R. Henderson, F. Marshall, A. Jazayeri and M. Weir for constructive comments on the manuscript.

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  1. Francesca Magnani, Maria J Serrano-Vega, Yoko Shibata, Saba Abdul-Hussein, Guillaume Lebon & Jennifer Miller-Gallacher

    Present address: Present addresses: Dipartimento di Biologia e Biotecnologie, Universita' di Pavia, Pavia, Italy (F.M.); Heptares Therapeutics, Welwyn Garden City, UK (M.J.S.-V.); CRUK MedImmune Alliance Laboratory, Great Abington, Cambridge, UK (Y.S.); Department of Biochemistry and Biophysics, University of Stockholm, Stockholm, Sweden (S.A.-H.); Institut de Génomique Fonctionnelle, Montpellier, France (G.L.); FIRS Laboratories, RSR Limited, Cardiff, UK (J.M.-G.).,

  2. Francesca Magnani, Maria J Serrano-Vega and Yoko Shibata: These authors contributed equally to the initial development of the methodology and are listed alphabetically.

Authors and Affiliations

  1. MRC Laboratory of Molecular Biology, Cambridge, UK

    Francesca Magnani, Maria J Serrano-Vega, Yoko Shibata, Saba Abdul-Hussein, Guillaume Lebon, Jennifer Miller-Gallacher, Ankita Singhal, Annette Strege, Jennifer A Thomas & Christopher G Tate

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All authors contributed to the development of techniques described in this paper. C.G.T. wrote the manuscript and coordinated contributions from all the other authors.

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Correspondence to Christopher G Tate.

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C.G.T. is a consultant for Heptares Therapeutics, and this work was funded partly by Pfizer and Heptares Therapeutics.

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Magnani, F., Serrano-Vega, M., Shibata, Y. et al. A mutagenesis and screening strategy to generate optimally thermostabilized membrane proteins for structural studies. Nat Protoc 11, 1554–1571 (2016). https://doi.org/10.1038/nprot.2016.088

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