ArticleNature protocols2016
A mutagenesis and screening strategy to generate optimally thermostabilized membrane proteins for structural studies.
Article in Nature protocols, 2016. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 50 papers.
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Who cites it
50 citing papers in PubMed.
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- Structural basis for CCR6 modulation by allosteric antagonists.Nature communications · 2024Article
- One-shot design elevates functional expression levels of a voltage-gated potassium channel.Protein science : a publication of the Protein Society · 2024Article
- Fluorescence-Detection Size-Exclusion Chromatography-Based Thermostability Assay for Membrane Proteins.Methods in molecular biology (Clifton, N.J.) · 2023Article
- eGFP as an All-in-One Tag for Purification of Membrane Proteins.Methods in molecular biology (Clifton, N.J.) · 2023Article
- Evolutionary analyses reveal immune cell receptor GPR84 as a conserved receptor for bacteria-derived molecules.iScience · 2022Article
- Intermolecular Interactions in G Protein-Coupled Receptor Allosteric Sites at the Membrane Interface from Molecular Dynamics Simulations and Quantum Chemical Calculations.Journal of chemical information and modeling · 2022Article
- Protein Design: From the Aspect of Water Solubility and Stability.Chemical reviews · 2022Review
- Membranes under the Magnetic Lens: A Dive into the Diverse World of Membrane Protein Structures Using Cryo-EM.Chemical reviews · 2022Review
- Detergents and alternatives in cryo-EM studies of membrane proteins.Acta biochimica et biophysica Sinica · 2022Review
- International Union of Basic and Clinical Pharmacology. CXII: Adenosine Receptors: A Further Update.Pharmacological reviews · 2022Review
- Structure and activity of a thermally stable mutant of Acanthamoeba actophorin.Acta crystallographica. Section F, Structural biology communications · 2022Article
- Universal platform for the generation of thermostabilized GPCRs that crystallize in LCP.Nature protocols · 2022Review
- Stabilization and structure determination of integral membrane proteins by termini restraining.Nature protocols · 2022Review
- Angiotensin and Endothelin Receptor Structures With Implications for Signaling Regulation and Pharmacological Targeting.Frontiers in endocrinology · 2022Review
- The high-throughput production of membrane proteins.Emerging topics in life sciences · 2021Article
- Structure determination of GPCRs: cryo-EM compared with X-ray crystallography.Biochemical Society transactions · 2021Review
- Agonists and allosteric modulators promote signaling from different metabotropic glutamate receptor 5 conformations.Cell reports · 2021Article
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10 authors.
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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 (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.
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