The temporal control afforded by the small-molecule labeling of genetically encoded protein tags, such as the HaloTag domain, has enabled a range of experiments that are not possible with standard fluorescent proteins. The need to wash away excess unreacted fluorescent label remains a limitation of this approach for intracellular labeling. We developed a novel molecular strategy for engendering a strong light-up signal in the fluorescence tagging of the HaloTag protein domain. Our approach relies on the properties of twisted intramolecular charge transfer (TICT) dyes, whose freely rotating bonds and internal donor-acceptor structure give them low quantum yields in solution. We designed a set of haloalkane-derivatized dyes having TICT structures potentially narrow enough to fit into the HaloTag domain’s binding pocket, which may restrict the bond rotation of the fluorophore. The best performing dye, with a chloroheptyl linker, yields a 27-fold fluorescence emission enhancement (λex = 535 nm, Em(max) = 616 nm) upon reaction with the HaloTag domain in vitro. We also prepared a chloroheptyl naphthalene-based dye for emission further to the red (Em(max) = 653 nm). These two dyes were successfully employed in the wash-free imaging of protein localization in E. coli, using a HaloTag fusion of the FtsZ protein. The dyes developed are easily-synthesized enzyme substrates with low background and large Stokes shifts, which offer substantial benefits over known fluorescent substrates for the HaloTag enzyme.

Fluorogenic dyes for haloalkane-based protein labeling in vitro and in bacterial cells

253rd National Meeting (2017)

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