Ratiometric Bioluminescent Zinc Sensor Proteins to Quantify Serum and Intracellular Free Zn(2+)

[Image: see text] Fluorescent Zn(2+) sensors play a pivotal role in zinc biology, but their application in complex media such as blood serum or plate reader-based cellular assays is hampered by autofluorescence and light scattering. Bioluminescent sensor proteins provide an attractive alternative to fluorescent sensors for these applications, but the only bioluminescent sensor protein developed so far, BLZinCh, has a limited sensor response and a suboptimal Zn(2+) affinity. In this work, we expanded the toolbox of bioluminescent Zn(2+) sensors by developing two new sensor families that show a large change in the emission ratio and cover a range of physiologically relevant Zn(2+) affinities. The LuZi platform relies on competitive complementation of split NanoLuc luciferase and displays a robust, 2-fold change in red-to-blue emission, allowing quantification of free Zn(2+) between 2 pM and 1 nM. The second platform was developed by replacing the long flexible GGS linker in the original BLZinCh sensor by rigid polyproline linkers, yielding a series of BLZinCh-Pro sensors with a 3–4-fold improved ratiometric response and physiologically relevant Zn(2+) affinities between 0.5 and 1 nM. Both the LuZi and BLZinCh-Pro sensors allowed the direct determination of low nM concentrations of free Zn(2+) in serum, providing an attractive alternative to more laborious and/or indirect approaches to measure serum zinc levels. Furthermore, the genetic encoding of the BLZinCh-Pro sensors allowed their use as intracellular sensors, where the sensor occupancy of 40–50% makes them ideally suited to monitor both increases and decreases in intracellular free Zn(2+) concentration in simple, plate reader-based measurements, without the need for fluorescence microscopy.