TY - JOUR T1 - High performance intensiometric direct- and inverse-response genetically encoded biosensors for citrate JF - bioRxiv DO - 10.1101/2020.04.12.038547 SP - 2020.04.12.038547 AU - Yufeng Zhao AU - Yi Shen AU - Yurong Wen AU - Robert E. Campbell Y1 - 2020/01/01 UR - http://biorxiv.org/content/early/2020/04/13/2020.04.12.038547.1.abstract N2 - 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/Fmin ~ 18) designated Citroff1 and a direct-response biosensor (ΔF/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.Competing Interest StatementThe authors have declared no competing interest. ER -