Engineering of mCherry variants with long Stokes shift, red-shifted fluorescence, and low cytotoxicity

MCherry, the Discosoma sp. mushroom coral-derived monomeric red fluorescent protein (RFP), is a commonly used genetically encoded fluorophore for live cell fluorescence imaging. We have used a combination of protein design and directed evolution to develop mCherry variants with low cytotoxicity to Escherichia coli and altered excitation and emission profiles. These efforts ultimately led to a long Stokes shift (LSS)-mCherry variant (λ(ex) = 460 nm and λ(em) = 610 nm) and a red-shifted (RDS)-mCherry variant (λ(ex) = 600 nm and λ(em) = 630 nm). These new RFPs provide insight into the influence of the chromophore environment on mCherry’s fluorescence properties, and may serve as templates for the future development of fluorescent probes for live cell imaging.