9HKF

X-Ray crystal structure of a photoswitchable HaloTag bound to JF635

Summary for 9HKF

• Entry DOI: 10.2210/pdb9hkf/pdb
• Descriptor: Haloalkane dehalogenase,non-specific serine/threonine protein kinase, CHLORIDE ION, FLAVIN MONONUCLEOTIDE, ... (6 entities in total)
• Functional Keywords: halotag, lov domain, fmn, dye, photoswitch, pshalotag, light-oxygen-voltage-sensing domain, rhodamine, biosynthetic protein
• Biological source: Rhodococcus rhodochrous; More
• Total number of polymer chains: 2
• Total formula weight: 103608.32

Authors

Weidenhausen, J.,Ugarte-Uribe, B.,Walterspiel, F.,Mueller, C.W.,Deo, C. (deposition date: 2024-12-03, release date: 2025-11-05)

Primary citation

Walterspiel, F.,Ugarte-Uribe, B.,Weidenhausen, J.,Vincent, M.,Narayanasamy, K.K.,Dimitriadi, A.,Khan, A.U.M.,Fritsch, M.,Muller, C.W.,Zimmermann, T.,Deo, C.
A Photoswitchable HaloTag for Spatiotemporal Control of Fluorescence in Living Cells.
Angew.Chem.Int.Ed.Engl., :e202424955-e202424955, 2025

PubMed Abstract: Photosensitive fluorophores, whose emission can be controlled using light, are essential for advanced biological imaging, enabling precise spatiotemporal tracking of molecular features and facilitating super-resolution microscopy techniques. 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 labeling methods. To address these limitations, we introduce chemigenetic photoswitchable fluorophores, leveraging the self-labeling HaloTag protein with fluorogenic rhodamine dye ligands. By incorporating a light-responsive protein domain into HaloTag, we engineer a tunable, photoswitchable HaloTag (psHaloTag), which can reversibly modulate the fluorescence of a bound dye-ligand via a light-induced conformational change. Our best performing psHaloTag variants show excellent performance in living cells, with large, reversible, deep-red fluorescence turn-on upon 450 nm illumination across various biomolecular targets and SMLM compatibility. Together, this work establishes the chemigenetic approach as a versatile platform for the design of photoswitchable reporters, tunable through both genetic and synthetic modifications, with promising applications for dynamic imaging.

PubMed: 41131894
DOI: 10.1002/anie.202424955

Experimental method

X-RAY DIFFRACTION (2.4 Å)