Chimeric Construct with mTagBFP2 and mCherry Is a Ratiometric Fluorescent Sensor for Nitric Oxide

Abstract

Although genetically encoded fluorescent sensors are fundamental tools to gain an understanding of intracellular nitric oxide (NO) dynamics, their development is still limited. We recently introduced the blue-emitting fluorescent protein (FP) mTagBFP2 as a NO sensor, where the chromophoric group formed upon S-nitrosylation of Cys residues leads to fluorescence quenching through excitation energy transfer. In this work, we further elaborated on this concept and devised a simple, two-domain, ratiometric NO sensor. mTagBFP2 was fused, with a suitable linker, to mCherry, a red-emitting fluorescent protein. As mCherry lacks cysteine residues, it is insensitive to NO and provides a convenient reference signal to normalize the response of mTagBFP2 fluorescence emission in a ratiometric detection. We demonstrate the capability of the construct to sense NO in the micromolar range in solution (Kd ca. 1 μM) and within mammalian (HeLa) and bacterial (Escherichia coli (E. coli)) cells.