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High-Performance Intensiometric Direct- and Inverse-Response Genetically Encoded Biosensors for Citrate
[Image: see text] Motivated by the growing recognition of citrate as a central metabolite in a variety of biological processes associated with healthy and diseased cellular states, we have developed a series of high-performance genetically encoded citrate biosensors suitable for imaging of citrate c...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Chemical Society
2020
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7453566/ https://www.ncbi.nlm.nih.gov/pubmed/32875085 http://dx.doi.org/10.1021/acscentsci.0c00518 |
Sumario: | [Image: see text] Motivated by the growing recognition of citrate as a central metabolite in a variety of biological processes associated with healthy and diseased cellular states, we have developed a series of high-performance genetically encoded citrate biosensors suitable for imaging of citrate concentrations in mammalian cells. The design of these biosensors was guided by structural studies of the citrate-responsive sensor histidine kinase and took advantage of the same conformational changes proposed to propagate from the binding domain to the catalytic domain. Following extensive engineering based on a combination of structure guided mutagenesis and directed evolution, we produced an inverse-response biosensor (ΔF/F(min) ≈ 18) designated Citroff1 and a direct-response biosensor (ΔF/F(min) ≈ 9) designated Citron1. We report the X-ray crystal structure of Citron1 and demonstrate the utility of both biosensors for qualitative and quantitative imaging of steady-state and pharmacologically perturbed citrate concentrations in live cells. |
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