{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,6,24]],"date-time":"2026-06-24T05:47:32Z","timestamp":1782280052497,"version":"3.54.5"},"posted":{"date-parts":[[2025,3,23]]},"group-title":"Biochemistry","reference-count":86,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,3,23]],"date-time":"2025-03-23T00:00:00Z","timestamp":1742688000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,3,23]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  The duality of nitrate is nowhere best exemplified than in human physiology \u2013 a detrimental pollutant but also a protective nutrient and signaling ion \u2013 particularly as connected to reactive nitrogen oxides. 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. However, imaging technologies for mammalian cell applications remain rare. Here, we set out to design and engineer a two-domain chimera fusing the split green fluorescent protein EGFP and the nitrate recognition domain NreA from\n                  <jats:italic>Staphylococcus carnosus<\/jats:italic>\n                  . Over 7 rounds of directed evolution, 15 mutations were accumulated resulting in the functional biosensor NitrOFF. NitrOFF has a high degree of allosteric communication between the domains reflected in a turn-off intensiometric response (\n                  <jats:italic>K<\/jats:italic>\n                  <jats:sub>d<\/jats:sub>\n                  \u2248 9 \u00b5M). This was further reinforced by X-ray crystal structures of apo and nitrate bound NitrOFF, which revealed that the two domains undergo a large-scale conformational rearrangement that changes the relative positioning of the EGFP and NreA domains by 68.4\u00b0. Such a dramatic difference was triggered by the formation of a long helix at the engineered linker connecting the two domains, peeling the \u03b27 strand off the EGFP and thus extinguishing the fluorescence upon nitrate binding. Finally, as a proof-of-concept, we highlighted the utility of this first-generation biosensor to monitor exogenous nitrate uptake and modulation in a human embryonic kidney (HEK) 293 cell line.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>Abstract Figure<\/jats:title>\n                  <jats:fig id=\"ufig1\" position=\"float\" fig-type=\"figure\" orientation=\"portrait\">\n                    <jats:graphic xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" xlink:href=\"644677v1_ufig1\" position=\"float\" orientation=\"portrait\"\/>\n                  <\/jats:fig>\n                <\/jats:sec>","DOI":"10.1101\/2025.03.22.644677","type":"posted-content","created":{"date-parts":[[2025,3,24]],"date-time":"2025-03-24T02:14:17Z","timestamp":1742782457000},"source":"Crossref","is-referenced-by-count":0,"title":["NitrOFF: An engineered fluorescent biosensor to illuminate nitrate transport in living cells"],"prefix":"10.64898","author":[{"given":"Mariah A.","family":"Cook","sequence":"first","affiliation":[{"name":"Department of Chemistry and Biochemistry, The University of Texas at Dallas, Richardson, TX 75080"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jonathan D.","family":"Smailys","sequence":"additional","affiliation":[{"name":"Department of Molecular Biosciences, University of Texas, Austin, TX 78712"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ke","family":"Ji","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Biochemistry, The University of Texas at Dallas, Richardson, TX 75080"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shelby M.","family":"Phelps","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Biochemistry, The University of Texas at Dallas, Richardson, TX 75080"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jasmine N.","family":"Tutol","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Biochemistry, The University of Texas at Dallas, Richardson, TX 75080"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wantae","family":"Kim","sequence":"additional","affiliation":[{"name":"McKetta Department of Chemical Engineering, University of Texas, Austin, TX 78712"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Whitney S. Y.","family":"Ong","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Biochemistry, The University of Texas at Dallas, Richardson, TX 75080"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Weicheng","family":"Peng","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Biochemistry, The University of Texas at Dallas, Richardson, TX 75080"},{"name":"Department of Biological Sciences, The University of Texas at Dallas, Richardson, TX 75080"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Caden","family":"Maydew","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Biochemistry, The University of Texas at Dallas, Richardson, TX 75080"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Y. Jessie","family":"Zhang","sequence":"additional","affiliation":[{"name":"Department of Molecular Biosciences, University of Texas, Austin, TX 78712"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sheel C.","family":"Dodani","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Biochemistry, The University of Texas at Dallas, Richardson, TX 75080"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026041200250483000_2025.03.22.644677v1.1","first-page":"73","article-title":"Nitrate: The Dr. Jekyll and Mr. Hyde of Human Health? 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