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| == A Photoswitchable HaloTag for Spatiotemporal Control of Fluorescence in Living Cells <ref> A Photoswitchable HaloTag for Spatiotemporal Control of Fluorescence in Living Cells https://doi.org/10.1002/anie.202424955</ref>==
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| <StructureSection load='9HKF' size='340' side='right' caption='Crystal structure of psHaloTag labeled with JF635-HTL in the dark state'>
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| 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. <ref> Photoswitchable Fluorescent Proteins: Ten Years of Colorful Chemistry and Exciting Applications https:// doi.org/10.1016/j.cbpa.2013.05.031</ref>. Many current systems are limited in tracking long-term, cyclical processes or in refreshing the pool of observable molecules.
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| 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.
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| ==== Features ====
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| # Photoswitching | Reversible ON/OFF fluorescence control using 450 nm light.
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| # Chemigenetic | Genetically encoded protein plus high-performance synthetic dye.
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| # Live-Cell Ready | Robust, visible-light activation across various targets.
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| ==== Mechanism ====
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| The psHaloTag works as an allosteric Photoswitch.
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| *Protein Scaffold: An engineered HaloTag protein with the light-sensing sAsLOV2 domain.
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| *Ligand: Binds to a <scene name='10/1096855/Puj/2'>fluorogenic rhodamine dye (JF635-HTL)</scene>
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| :Light ON (450 nm): Light activates the sAsLOV2 domain using <scene name='10/1096855/Fmn/1'>Flavin Mononucleotide (FMN)</scene>
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| cofactor, causing a conformational change (Jα helix unfolding).
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| :Fluorescence ON: This change forces the bound dye into its fluorescent state.
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| :Light OFF (Dark): The protein structure relaxes back to its original shape, shifting
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| the dye back to its non-fluorescent state.
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| <scene name='10/1096855/Cl_ion/1'>Cl</scene>
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| <scene name='10/1096855/Gol/3'>GOL</scene>
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| [[Image:Principle of HaloTag.jpg | frame | upright= 1.5 |none | alt= | Fig.1. General principle of photoswitchable HaloTag (psHaloTag) and open-closed equilibrium of JF635-HaloTag ligand (HTL). L1 and L2 denote linkers. ]]
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| 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.
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| ==== Structural highlights ====
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| 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.
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| ==== Relevance ====
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| 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.
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| 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.
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| 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).
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| ====References====
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| <references/>
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