A procedure used to inactivate specific proteins with high spatial and temporal precision. The chromophore is illuminated with a strong light, generating short-lived reactive oxygen species that inactivate proteins in its vicinity. There are genetically coded CALI that can cause protein inactivation using widefield illumination with no lasers required.
A photoactivatable engineered variant of Green Fluorescent Protein (GFP) used to study calcium levels in cells. Cameleon is a sensor protein that contains a calmodulin (CALcium MODULated proteIN) component. When calcium binds to this component, it undergoes a conformational change leading to emission of light at an altered wavelength. The first cameleon was created using Blue Fluorescent Protein (BFP), Green Fluorescent Protein, calmodulin and a calmodulin-binding peptide. When Calcium binds to this calmodulin-binding peptide, it brings the two otherwise separated BFP and GFP in close proximity to one another, increasing their FRET efficiency.
Joining the family of photoactivated light-sensitive compounds are channelrhodopsins. These are a family of opsin proteins that function as light-gated ion channels. Use of channelrhodopsins in living cells enables control of ion influx, intracellular activity and other cellular processes in response to fluorescence illumination. ChR2 channelrhodopsin absorbs light at 480nm and a conformational change opens the channel to allow ion influxes. These can also be fused to fluorescence proteins and altered to open and close when exposed to a specific wavelengths of light. They can be expressed in cells through several transfection techniques for applications such as depolarization of neuronal cells, neuron photostimulation or optogenetics.
Kaede protein is a photoactivatable fluorescent protein naturally originated from a stony coral, Trachyphyllia geoffroyi. When irradiated with UV or violet light (350-400 nm), Kaede undergoes irreversible photoconversion from green to red fluorescence. Kaede's tetramerization makes it unlikely to aggregate when fused to other proteins. In its unactivated state, when excited at 480nm, it emits fluorescence with a peak at 519nm. When activated with UV light, its major absorption peak is red shifted to 570nm. Kaede can be used as an optical marker for protein labeling and gene expression to study cell behavior. It is particularly useful in providing a way to view neurons individually. Photoactivating one neuron in relation to another can help separate the neuronal processes by color.
Photoactivatable GFP (green fluorescent protein) is a variant of GFP. As described in the XFP application section, GFP has a major excitation peak at a 395 nm and a minor one at 475 nm. Intense illumination of the variant PA-GFP at 400nm shifts the peak absorbance to 475nm, causing a 100-fold increase in green fluorescence when excited at 488 nm. PA-GFP offers a new way of studying intracellular protein dynamics by tracking only those photoactivated molecules that are present and visible within the cell. Following activation, there is an increase in fluorescence intensity at the activation site. The redistribution of these molecules can be studied using time-lapse imaging.
Caged compounds are biologically inactive substances that contain a photoactivatable group. Uncaging occurs upon absorption of an appropriate photon, causing cleavage of the caged group which releases the active biological substance. This makes it possible to study events that are dependent on exposure to a certain chain of events without need of all the events. This technique enables precise spatial and temporal control of activation of a signal molecule, providing answers to neurological questions and even drug delivery methods.
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