Abstract
Genetically encoded photosensitizers, proteins that produce reactive oxygen species when illuminated with visible light, are increasingly used as optogenetic tools. Their applications range from ablation of specific cell populations to precise optical inactivation of cellular proteins. Here, we report an orange mutant of red fluorescent protein KillerRed that becomes toxic when illuminated with blue or green light. This new protein, KillerOrange, carries a tryptophan-based chromophore that is novel for photosensitizers. We show that KillerOrange can be used simultaneously and independently from KillerRed in both bacterial and mammalian cells offering chromatic orthogonality for light-activated toxicity.
Highlights
Fluorescent proteins of GFP family are widely used in molecular and cellular biology as passive reporters of the cellular structures and processes [1]
The structure of beta-barrel maintains a path through the protein that reaches the chromophore and is accessible by the water and oxygen molecules [4,5]. This unusual example belongs to the red fluorescent protein KillerRed developed from the jellyfish chromoprotein anm2CP by directed evolution [6]
In order to develop a non-red mutant we first performed a round of random mutagenesis on KillerRed gene expecting to find a substitution that disrupts acylimine group formation during the chromophore maturation
Summary
Fluorescent proteins of GFP family are widely used in molecular and cellular biology as passive reporters of the cellular structures and processes [1]. The chromophore of a fluorescent protein is buried deeply inside a protein beta-barrel and shielded from the surrounding solvent. This is believed to explain a much lower level of phototoxicity of a typical fluorescent protein comparing to small organic dyes [2,3]. The structure of beta-barrel maintains a path through the protein that reaches the chromophore and is accessible by the water and oxygen molecules [4,5] This unusual example belongs to the red fluorescent protein KillerRed developed from the jellyfish chromoprotein anm2CP by directed evolution [6]. When illuminated with green or orange light, KillerRed produces reactive oxygen species and demonstrates three orders of magnitude higher level of phototoxicity than other fluorescent proteins [6]
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