The Photoswitchable Halo Tag (psHaloTag) is a unique chemigenetic system designed to provide reversible, light-controlled fluorescence tunable through both genetic and synthetic modifications, with promising applications for dynamic imaging in biological microscopy [1]. psHaloTag addresses a major gap in the availability of reliable, reversible systems suitable for live-cell imaging. Many current systems are limited in tracking long-term, cyclical processes or in refreshing the pool of observable molecules. psHaloTag combines the genetically encoded HaloTag protein with the light-sensing sAsLOV2 domain. When illuminated with 450 nm light, psHaloTag undergoes a reversible change that activates a bound rhodamine dye ligand, resulting in a significant increase in fluorescence. This strong, multiple-cycle reporter overcomes the limitations of irreversible systems. It is an important tool for achieving precise spatiotemporal control in demanding applications such as live-cell Super-Resolution Microscopy (SMLM), where many established photosensitive probes cannot be reactivated.
Function
The psHaloTag works as an allosteric photoswitch.
- Protein Scaffold: An engineered HaloTag protein with the light-sensing sAsLOV2 domain.
- Ligand: Binds to a fluorogenic rhodamine dye (e.g., JF635-HTL).
- Light ON (450 nm): Light activates the sAsLOV2 domain using FMN cofactor, causing a conformational change (Jα helix unfolding).
- Fluorescence ON: This change forces the bound dye into its fluorescent state.
- Light OFF (Dark): The protein structure relaxes back to its original shape, shifting the dye back to its non-fluorescent state.
Mechanism
Structural highlights
Relevance
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