NitrOFF: An Engineered Fluorescent Biosensor to Illuminate Nitrate Transport in Living Cells.

ABSTRACT: The duality of nitrate is nowhere better exemplified than in human physiology-a detrimental pollutant but also a protective nutrient-particularly as connected to nitric oxide. Aside from limited insights into nitrate uptake and storage, foundational nitrate biology has lagged. Genetically encoded fluorescent biosensors can address this gap with real-time imaging, but such technologies for mammalian cell applications remain rare. Here, we designed and engineered a biosensor fusing the green fluorescent protein EGFP and the nitrate recognition domain NreA from Staphylococcus carnosus. Seven rounds of directed evolution and 15 mutations resulted in NitrOFF. NitrOFF has a high degree of allosteric communication between the domains reflected in a turn-off intensiometric response (K_d

SUBMITTER: Cook MA 

PROVIDER: S-EPMC12440377 | biostudies-literature | 2025 Aug

REPOSITORIES: biostudies-literature