A Chemigenetic Ca2+ Indicator Based on a Red Fluorescent Protein and a Synthetic Chelator
Abstract Fluorescent indicators are indispensable imaging tools for visualizing the spatiotemporal dynamics of biological processes. Red fluorescent indicators are in particularly high demand because they offer compatibility with existing green fluorescent indicators or optogenetic tools, and longer wavelength fluorescence has inherent advantages for biological applications. We previously described a chemigenetic indicator design that combines a green fluorescent protein and a synthetic chelator in an effort to combine the advantages of conventional protein-based biosensors and synthetic chemosensors. We now demonstrate that this chemigenetic design can be extended to red fluorescent proteins. Through screening of variants with a range of chromophore–chelator orientations, followed by directed evolution, we developed a red fluorescent calcium ion (Ca2+) indicator with 5.4-fold fluorescence intensity change when going from 0 to 39 μM Ca2+ with purified proteins. Although the functionality of the current version is lost when expressed in mammalian cells and the selectivity is low, these results establish this chemigenetic design as a strategy that can be extended to other fluorescent protein color variants.