User:Rupal Yadav; BI3323-Aug25.

Reversibly photoswitchable fluorescent proteins (RSFPs) are fluorescent proteins whose fluorescence, upon excitation at a certain wavelength, can be switched on or off by light in a reversible manner. ^[2] Many current systems are limited in tracking long-term, cyclical processes or in refreshing the pool of observable molecules.

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. psHaloTag addresses a major gap in the availability of reliable, reversible systems suitable for live-cell imaging. 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.

Features

1. Photoswitching | Reversible ON/OFF fluorescence control using 450 nm light.
2. Chemigenetic | Genetically encoded protein plus high-performance synthetic dye.
3. 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

Light ON (450 nm): Light activates the sAsLOV2 domain using , 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.

Fig.1. General principle of photoswitchable HaloTag (psHaloTag) and open-closed equilibrium of JF635-HaloTag ligand (HTL). L1 and L2 denote linkers.

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 stabilise 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 colors and stability in light whose performance does not rely on oxygen and is less affected by pH changes compared to traditional fluorescent proteins (FPs).

References

1. ↑ A Photoswitchable HaloTag for Spatiotemporal Control of Fluorescence in Living Cells https://doi.org/10.1002/anie.202424955
2. ↑ Photoswitchable Fluorescent Proteins: Ten Years of Colorful Chemistry and Exciting Applications https:// doi.org/10.1016/j.cbpa.2013.05.031