Redesign of Calmodulin for Genetically Encoded Zn²⁺ Sensing with High Dynamic Range

The development of genetically encoded Zn²⁺ probes (GEZPs) has been hindered by the limited signal transduction efficiency of natural zinc-binding domains, but the encoded Ca²⁺ indicators utilize the cooperative conformational changes of calmodulin (CaM) to achieve superior dynamic ranges. Here, we rationally redesigned the calcium-binding protein CaM in Ca²⁺ indicators to develop a biosensor, mNG-ZnM1.0, which shows a high dynamic range and selectivity for Zn²⁺. Specifically, by utilizing Zn²⁺ coordination properties, key Ca²⁺-binding sites in the four EF-hand motifs of CaM were mutated to cysteine or histidine. This yielded the 20H variant (mNG-ZnM), which responded to Zn²⁺ but was insensitive to Ca²⁺. However, it lost the ability to generate a high dynamic range response. Considering the high conservation of the first coordinating residue (Asp1) in each EF-hand motif, saturation mutagenesis identified Asp1 as the critical residue for achieving high dynamic range Zn²⁺ sensing in mNG-ZnM. Subsequent directed evolution optimization produced mNG-ZnM1.0, which exhibited significantly enhanced Zn²⁺ response. The dynamic range of mNG-ZnM1.0 for Zn²⁺ detection reached up to 20-fold in vitro and 4-fold in live cells (F_max/F_min), performing comparably or superiorly to traditional GEZPs. Using zebrafish expressing mNG-ZnM1.0, we successfully visualized the Zn²⁺ dynamics during development in vivo. Altogether, this study expands the repertoire of sensing modules for GEZPs and provides a paradigm for reprogramming classical protein scaffolds to enable additional functionalities.