Cook Mariah A, Smailys Jonathan D, Ji Ke, Phelps Shelby M, Tutol Jasmine N, Kim Wantae, Ong Whitney S Y, Peng Weicheng, Maydew Caden, Zhang Y Jessie, Dodani Sheel C
Department of Chemistry and Biochemistry, The University of Texas at Dallas, 800 West Campbell Road, Richardson, TX, 75080, USA.
Department of Molecular Biosciences, University of Texas, 100 East 24th Street, Austin, TX, 78712, USA.
Angew Chem Int Ed Engl. 2025 Aug 26:e202508058. doi: 10.1002/anie.202508058.
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 ≈ 9 µM). This was further reinforced by X-ray crystal structures of apo and nitrate-bound NitrOFF, which revealed a large-scale conformational rearrangement changing the relative positioning of the domains by 68.4°. This dramatic difference was triggered by the formation of a long helix at the engineered linker connecting the two domains, peeling the β7 strand off the EGFP and thus extinguishing the fluorescence upon nitrate binding. Finally, we highlighted the utility of NitrOFF to monitor exogenous nitrate uptake and modulation in the human embryonic kidney (HEK) 293 cell line.
硝酸盐的双重性在人体生理学中体现得淋漓尽致——它是一种有害污染物，但也是一种保护性营养素，尤其是与一氧化氮相关时。除了对硝酸盐摄取和储存的有限认识外，基础硝酸盐生物学一直滞后。基因编码的荧光生物传感器可以通过实时成像来填补这一空白，但用于哺乳动物细胞应用的此类技术仍然很少。在这里，我们设计并构建了一种生物传感器，它融合了绿色荧光蛋白EGFP和来自肉葡萄球菌的硝酸盐识别结构域NreA。经过七轮定向进化和15个突变，得到了NitrOFF。NitrOFF在各结构域之间具有高度的变构通讯，这反映在关闭型强度响应中（K≈9µM）。脱辅基和结合硝酸盐的NitrOFF的X射线晶体结构进一步证实了这一点，该结构揭示了一种大规模的构象重排，使各结构域的相对位置改变了68.4°。这种显著差异是由连接两个结构域的工程化连接子处形成的长螺旋引发的，该螺旋将β7链从EGFP上剥离，从而在硝酸盐结合时熄灭荧光。最后，我们强调了NitrOFF在监测人胚肾（HEK）293细胞系中外源硝酸盐摄取和调节方面的实用性。
