:Light OFF (Dark): The protein structure relaxes back to its original shape, shifting the dye back to its non-fluorescent state.
:Light OFF (Dark): The protein structure relaxes back to its original shape, shifting the dye back to its non-fluorescent state.
[[Image:Principle of HaloTag.jpg | thumb ]
[[Image:Principle of HaloTag.jpg | thumb ]]
The psHaloTag variants show a strong, reversible, deep-red fluorescence turn-on when activated by light. This provides a well-controlled tool for improved imaging.
The psHaloTag variants show a strong, reversible, deep-red fluorescence turn-on when activated by light. This provides a well-controlled tool for improved imaging.
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.
Photoswitching | Reversible ON/OFF fluorescence control using 450 nm light.
Chemigenetic | Genetically encoded protein plus high-performance synthetic dye.
Live-Cell Ready | Robust, visible-light activation across various targets.
Mechanism
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 (JF635-HTL).
Light ON (450 nm): Light activates the sAsLOV2 domain using Flavin Mononucleotide (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.
The psHaloTag variants show a strong, reversible, deep-red fluorescence turn-on when activated by light. This provides a well-controlled tool for improved imaging.
Structural highlights
The protein is made by inserting the light-sensing sAsLOV2 domain into the HaloTag protein. An extended helix connects the domains. This connection makes sure the light-induced structural change from the LOV domain is effectively transmitted to the HaloTag region, where the rhodamine dye is bound. Performance-boosting mutations are found near the headgroup of the rhodamine dye. These mutations likely stabilize the interaction to enhance the fluorescence ON/OFF ratio.
Relevance
Although irreversibly photoactivatable fluorophores are well established, reversible reporters that can be reactivated multiple times remain scarce, and only a few have been applied in living cells using generalizable protein labelling methods.
This system improves techniques like SMLM by allowing precise control of single-molecule emitter density over time by achieving sub-diffraction resolution in living cells.
Also, it exploits high-quality synthetic dyes for bright colours and stability in light whose performance does not rely on oxygen and is less affected by pH changes compared to traditional fluorescent proteins (FPs).
This is a sample scene created with SAT to color by Group, and another to make a transparent representation of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.
