Development of an Optical Zn²⁺ Probe Based on a Single Fluorescent Protein

Various fluorescent probes have been developed to reveal the biological functions of intracellular labile Zn²⁺. Here, we present Green Zinc Probe (GZnP), a novel genetically encoded Zn²⁺ sensor design based on a single fluorescent protein (single-FP). The GZnP sensor is generated by attaching two zinc fingers (ZF) of the transcription factor Zap1 (ZF1 and ZF2) to the two ends of a circularly permuted green fluorescent protein (cpGFP). Formation of ZF folds induces interaction between the two ZFs, which induces a change in the cpGFP conformation, leading to an increase in fluorescence. A small sensor library is created to include mutations in the ZFs, cpGFP and linkers between ZF and cpGFP to improve signal stability, sensor brightness and dynamic range based on rational protein engineering, and computational design by Rosetta. Using a cell-based library screen, we identify sensor GZnP1, which demonstrates a stable maximum signal, decent brightness (QY = 0.42 at apo state), as well as specific and sensitive response to Zn²⁺ in HeLa cells (F_max/F_min = 2.6, K_d = 58 pM, pH 7.4). The subcellular localizing sensors mito-GZnP1 (in mitochondria matrix) and Lck-GZnP1 (on plasma membrane) display sensitivity to Zn²⁺ (F_max/F_min = 2.2). This sensor design provides freedom to be used in combination with other optical indicators and optogenetic tools for simultaneous imaging and advancing our understanding of cellular Zn²⁺ function.