High-Performance Intensiometric Direct- and Inverse-Response Genetically Encoded Biosensors for Citrate

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/F-min approximate to 18) designated Citroffl and a direct- response biosensor (Delta F/F-min approximate to 9) designated Citronl. We report the X-ray crystal structure of Citronl and demonstrate the utility of both biosensors for qualitative and quantitative imaging of steady-state and pharmacologically perturbed citrate concentrations in live cells.