ArticleBiochimica et biophysica acta2013
Optimising the combination of thermostabilising mutations in the neurotensin receptor for structure determination.
Article in Biochimica et biophysica acta, 2013. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 22 papers.
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Who cites it
22 citing papers in PubMed, 40 citations in OpenAlex.
- Engineering G protein-coupled receptors for stabilization.Protein science : a publication of the Protein Society · 2024Article
- One-shot design elevates functional expression levels of a voltage-gated potassium channel.Protein science : a publication of the Protein Society · 2024Article
- Engineering of Challenging G Protein-Coupled Receptors for Structure Determination and Biophysical Studies.Molecules (Basel, Switzerland) · 2021Review
- IMPROvER: the Integral Membrane Protein Stability Selector.Scientific reports · 2020Article
- Structural stability of purified human CFTR is systematically improved by mutations in nucleotide binding domain 1.Biochimica et biophysica acta. Biomembranes · 2018Article
- New approaches towards the understanding of integral membrane proteins: A structural perspective on G protein-coupled receptors.Protein science : a publication of the Protein Society · 2017Review
- A mutagenesis and screening strategy to generate optimally thermostabilized membrane proteins for structural studies.Nature protocols · 2016Article
- A generic selection system for improved expression and thermostability of G protein-coupled receptors by directed evolution.Scientific reports · 2016Article
- How Can Mutations Thermostabilize G-Protein-Coupled Receptors?Trends in pharmacological sciences · 2016Review
- Article
- Rapid Bioinformatic Identification of Thermostabilizing Mutations.Biophysical journal · 2015Article
- Identifying Thermostabilizing Mutations in Membrane Proteins by Bioinformatics.Biophysical journal · 2015Article
- GPCR structure, function, drug discovery and crystallography: report from Academia-Industry International Conference (UK Royal Society) Chicheley Hall, 1-2 September 2014.Naunyn-Schmiedeberg's archives of pharmacology · 2015Article
- Structural prerequisites for G-protein activation by the neurotensin receptor.Nature communications · 2015Article
- Structural dynamics and thermostabilization of neurotensin receptor 1.The journal of physical chemistry. B · 2015Article
- Stabilization of G protein-coupled receptors by point mutations.Frontiers in pharmacology · 2015Review
- Experimental phasing for structure determination using membrane-protein crystals grown by the lipid cubic phase method.Acta crystallographica. Section D, Biological crystallography · 2015Article
- Quality control in eukaryotic membrane protein overproduction.Journal of molecular biology · 2014Article
- Rapid Computational Prediction of Thermostabilizing Mutations for G Protein-Coupled Receptors.Journal of chemical theory and computation · 2014Article
- Article
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Authors and funding
7 authors at 3 institutions in 2 countries.
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Abstract
Conformational thermostabilisation of G protein-coupled receptors is a successful approach for their structure determination. We have recently determined the structure of a thermostabilised neurotensin receptor NTS1 in complex with its peptide agonist and here we describe the strategy for the identification and combination of the 6 thermostabilising mutations essential for crystallisation. First, thermostability assays were performed on a panel of 340 detergent-solubilised Ala/Leu NTS1 mutants and the best 16 thermostabilising mutations were identified. These mutations were combined pair-wise in nearly all combinations (119 out of a possible 120 combinations) and each mutant was expressed and its thermostability was experimentally determined. A theoretical stability score was calculated from the sum of the stabilities measured for each double mutant and applied to develop 24 triple mutants, which in turn led to the construction of 14 quadruple mutants. Use of the thermostability data for the double mutants to predict further mutant combinations resulted in a greater percentage of the triple and quadruple mutants showing improved thermostability than if only the thermostability data for the single mutations was considered. The best quadruple mutant (NTS1-Nag36k) was further improved by including an additional 2 mutations (resulting in NTS1-GW5) that were identified from a complete Ala/Leu scan of Nag36k by testing the thermostability of the mutants in situ in whole bacteria. NTS1-GW5 had excellent stability in short chain detergents and could be readily purified as a homogenous sample that ultimately allowed crystallisation and structure determination.
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