Abstract
Genetically encodable photosensitizers based on fluorescent proteins produce reactive oxygen species when illuminated with light. Although widely used as optogenetic tools, existing photosensitizers with green fluorescence possess suboptimal properties motivating for a search of new protein variants with efficient chromophore maturation and high phototoxicity. Here we report a mutant of the phototoxic fluorescent protein KillerRed protein with fluorescence in the green part of the spectrum. The mutant variant carries mutations I64L, D114G, and T115S and does not form a DsRed-like chromophore. The protein can be used as a template to create new genetically encodable photosensitizers that are spectrally different from KillerRed.
Highlights
Encodable photosensitizers based on fluorescent proteins produce reactive oxygen species when illuminated with light
We visually screened bacterial colonies exposed by 400 nm and 480 nm light to identify mutants with significant green fluorescence and identified the KillerRed I64L/ D114G/T115S mutant
Observed spectral properties indicated that the protein formed the “classical” GFP-like chromophore instead of the DsRed-like chromophore found in the parental KillerRed
Summary
Encodable photosensitizers based on fluorescent proteins produce reactive oxygen species when illuminated with light. Fluorescent proteins are widely used as genetically encodable tags for optical labeling of living systems [1] Their chromophores are located inside the protein structure and are protected from the surrounding solvent; most of the existing fluorescent proteins are passive reporter molecules: irradiation with light does not significantly affect cells expressing these markers. Members of the family produce reactive oxygen species that can damage the cell [2] Structural studies of these proteins have identified a water-filled channel that connects the chromophore to the solvent. Depending on the cellular localization and the excitation light dose, reactive oxygen species generated by KillerRed can lead to various physiological consequences — from inactivation of fusion proteins [2], to cell division arrest [5, 6] or cellular death through necrosis or apoptosis [2, 7]. ОРИГИНАЛЬНОЕ ИССЛЕДОВАНИЕ ГЕНЕТИКА has been used as a photosensitizer for treatment of tumors in model systems [7,8,9]
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