7OND

HaloTag Engineering for Enhanced Fluorogenicity and Kinetics with a Styrylpyridine Dye

Summary for 7OND

• Entry DOI: 10.2210/pdb7ond/pdb
• Descriptor: Haloalkane dehalogenase, 4-[(E)-2-[1-(7-chloranylheptyl)pyridin-1-ium-4-yl]ethenyl]-N,N-dimethyl-aniline, CHLORIDE ION, ... (5 entities in total)
• Functional Keywords: halotag, fluorescence, imaging, mammalian cells, fluorescent reporter, channel dye, directed evolution, fluorescent protein
• Biological source: Rhodococcus sp.
• Total number of polymer chains: 2
• Total formula weight: 69643.57

Authors

Stein, A.,Liang, A.D. (deposition date: 2021-05-25, release date: 2021-07-21, Last modification date: 2024-10-23)

Primary citation

Miro-Vinyals, C.,Stein, A.,Fischer, S.,Ward, T.R.,Deliz Liang, A.
HaloTag Engineering for Enhanced Fluorogenicity and Kinetics with a Styrylpyridium Dye.
Chembiochem, 22:3398-3401, 2021

PubMed Abstract: HaloTag is a small self-labeling protein that is frequently used for creating fluorescent reporters in living cells. The small-molecule dyes used with HaloTag are almost exclusively based on rhodamine scaffolds, which are often expensive and challenging to synthesize. Herein, we report the engineering of HaloTag for use with a chemically accessible, inexpensive fluorophore based on the dimethylamino-styrylpyridium dye. Through directed evolution, the maximum fluorogenicity and the apparent second-order bioconjugation rate constants could be improved up to 4-fold and 42-fold, respectively. One of the top variants, HT-SP5, enabled reliable imaging in mammalian cells, with a 113-fold fluorescence enhancement over the parent protein. Additionally, crystallographic characterization of selected mutants suggests the chemical origin of the fluorescent enhancement. The improved dye system offers a valuable tool for imaging and illustrates the viability of engineering self-labeling proteins for alternative fluorophores.

PubMed: 34609782
DOI: 10.1002/cbic.202100424

Experimental method

X-RAY DIFFRACTION (1.45 Å)