A novel molecular strategy for accelerating hydrazone and oxime formations, using bifunctional buffer compounds that not only control pH but also catalyze the reaction, is presented.

We describe a novel molecular strategy for engendering a strong light-up signal in fluorescence tagging of the genetically encoded HaloTag protein domain. We designed a set of haloalkane-derivatized dyes having twisted internal charge transfer (TICT) structures potentially narrow enough to partially fit into the enzyme’s haloalkane-binding channel. Testing a range of short chain lengths revealed a number of active dyes, with seven carbons yielding optimum light-up signal. The dimethylaminostilbazolium chloroheptyl dye (1d) yields a 27-fold fluorescence emission enhancement (λex = 535 nm; Em(max) = 616 nm) upon reaction with the protein. The control compound with standard 12-atom linkage shows less efficient signaling, consistent with our channel-binding hypothesis. For emission further to the red, we also prepared a chloroheptyl naphthalene-based dye; compound 2 emits at 653 nm with strong fluorescence enhancement upon reaction with the HaloTag domain. The two dyes (1d, 2) were successfully tested in wash-free imaging of protein localization in bacteria, using a HaloTag fusion of the filamenting temperature-sensitive mutant Z (FtsZ) protein in Escherichia coli (E. coli). The new dye conjugates are inexpensive and easily synthesized enzyme substrates with low background and large Stokes shifts, offering substantial benefits over known fluorescent substrates for the HaloTag enzyme.

HaloTag labeling technology has introduced unrivaled potential in protein chemistry and molecular and cellular biology. A wide variety of ligands have been developed to meet the specific needs of diverse applications, but only a single protein tag, DhaAHT, is routinely used for their incorporation. Following a systematic kinetic and computational analysis of different reporters, a tetramethylrhodamine- and three 4-stilbazolium-based fluorescent ligands, we showed that the mechanism of incorporating different ligands depends both on the binding step and the efficiency of the chemical reaction. By studying the different haloalkane dehalogenases DhaA, LinB, and DmmA, we found that the architecture of the access tunnels is critical for the kinetics of both steps and the ligand specificity. We showed that highly efficient labeling with specific ligands is achievable with natural dehalogenases. We propose a simple protocol for selecting the optimal protein tag for a specific ligand from the wide pool of available enzymes with diverse access tunnel architectures. The application of this protocol eliminates the need for expensive and laborious protein engineering.