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bioRxiv · 10.1101/2020.04.12.038547

High performance intensiometric direct- and inverse-response genetically encoded biosensors for citrate

Zhao, Y.·Wen, Y.·Shen, Y.·Campbell, R. E.
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Abstract

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 ({Delta}F/Fmin ~ 18) designated Citroff1 and a direct-response biosensor ({Delta}F/Fmin ~ 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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Zhao, Y., Wen, Y., Shen, Y., Campbell, R. E.. 2020-04-13. High performance intensiometric direct- and inverse-response genetically encoded biosensors for citrate. https://doi.org/10.1101/2020.04.12.038547

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