The two key substitutions in the chromophore environment of mKate2 to produce an enhanced FusionRed-like red fluorescent protein
Red fluorescent proteins (RFPs) are often probes of choice for living tissue microscopy and whole-body imaging. When choosing a specific RFP variant, the priority may be focused on fluorescence brightness, maturation rate, monomericity, excitation/emission wavelengths, and low toxicity, which are rarely combined in optimal way in a single protein. If the additional requirements such as prolonged fluorescence lifetime and/or blinking ability are applied, the available probes repertoire could become surprisingly narrow. Since the whole diversity of the conventional single-component RFPs belongs to just a few phylogenetic lines (with DsRed-, eqFP578- and eqFP611-derived being the major ones), it is not unexpected that their advantageous properties are split between close homologs. In such cases, a systematic mutagenetic analysis focused on variant-specific amino acid residues can shed light on the origins of sibling RFPs distinctness and might be beneficial for consolidation of their strengths in new RFP variants. For instance, the protein FusionRed, although being efficient in the fluorescence labeling due its good monomericity and low cytotoxicity, has undergone a considerable loss in fluorescence brightness/lifetime compared to the parental mKate2. In this contribution, we describe a fast-maturing monomeric RFP designed semi-rationally based on the mKate2 and FusionRed templates, outperforming both its parents in molecular brightness, having extended fluorescence lifetime, and showing spontaneous blinking pattern promising for nanoscopy use.