- Light-induced production of reactive oxygen species
- Direct expression in cells, easy targeting to various subcellular compartments
- No exogenous chemical compounds required for chromophore maturation
- Not toxic before activation by light irradiation
- Recommended for selective light-induced protein inactivation and cell killing
They can be used for precise inactivation of selected proteins in chromophore-assisted light inactivation (CALI) technique and for the light-induced cell killing. Besides KillerRed protein (and its derivative KillerOrange), all known to date photosensitizers require chemical compounds that should be introduced into living systems exogenously. KillerRed is the first genetically-encoded photosensitizer. Unlike chemical analogs, KillerRed can be directly expressed by target cells, both individually and in fusion with a target protein. It shows no cell toxic effects before light activation. Upon green or orange light irradiation, KillerRed generates ROS that damage the neighboring molecules.
KillerOrange was shown to be phototoxic to E.coli after illumination with blue or green (450-540 nm) light.
The blue-shifted spectrum of KillerOrange makes it potentially more suitable for two-photon microscopy than the parental KillerRed. Also, the large Stokes shift of over 40 nm should make it possible to spectrally separate signals of KillerOrange from cyan and green fluorescent proteins when the proteins are excited simultaneously by blue light. One can thus use green and cyan indicators to observe the effects of phototoxicity in real time without the need to change the excitation light.
KillerOrange-KillerRed pair can potentially be used to independently ablate two cell populations. This pair also promises the orthogonal optical control of the propagation of signaling cascades either by chromophore-assisted light inactivation of the participating proteins or by triggering cascades with hydrogen peroxide produced by KillerRed and likely by KillerOrange. KillerOrange-KillerRed tandem fusions or combination of various photosensitizers in one cassette may enhance phototoxicity under white light irradiation and may be useful as a research tool in biology.