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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...

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Detalles Bibliográficos
Autores principales: Zhao, Yufeng, Shen, Yi, Wen, Yurong, Campbell, Robert E.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Chemical Society 2020
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
Descripción
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.