RESUMEN
Hydrazone and oxime bond formation between α-nucleophiles (e.g. hydrazines, alkoxy-amines) and carbonyl compounds (aldehydes and ketones) is convenient and is widely applied in multiple fields of research. While the reactants are simple, a substantial drawback is the relatively slow reaction at neutral pH. Here we describe a novel molecular strategy for accelerating these reactions, using bifunctional buffer compounds that not only control pH but also catalyze the reaction. The buffers can be employed at pH 5-9 (5-50 mM) and accelerate reactions by several orders of magnitude, yielding second-order rate constants of >10 M-1 s-1. Effective bifunctional amines include 2-(aminomethyl)imidazoles and N,N-dimethylethylenediamine. Unlike previous diaminobenzene catalysts, the new buffer amines are found to have low toxicity to human cells, and can be used to promote reactions in cellular applications.
RESUMEN
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.
Asunto(s)
Proteínas Bacterianas/química , Proteínas del Citoesqueleto/química , Colorantes Fluorescentes/química , Ingeniería de Proteínas/métodos , Proteínas Recombinantes de Fusión/química , Bacterias/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Colorantes Fluorescentes/síntesis química , Glutatión Transferasa/química , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Hidrolasas/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Espectrometría de FluorescenciaRESUMEN
Genetically encoded methods for protein conjugation are of high importance as biological tools. Here we describe the development of a new class of dyes for genetically encoded tagging that add new capabilities for protein reporting and detection via HaloTag methodology. Oligodeoxyfluorosides (ODFs) are short DNA-like oligomers in which the natural nucleic acid bases are replaced by interacting fluorescent chromophores, yielding a broad range of emission colors using a single excitation wavelength. We describe the development of an alkyl halide dehalogenase-compatible chloroalkane linker phosphoramidite derivative that enables the rapid automated synthesis of many possible dyes for protein conjugation. Experiments to test the enzymatic self-conjugation of nine different DNA-like dyes to proteins with HaloTag domains in vitro were performed, and the data confirmed the rapid and efficient covalent labeling of the proteins. Notably, a number of the ODF dyes were found to increase in brightness or change color upon protein conjugation. Tests in mammalian cellular settings revealed that the dyes are functional in multiple cellular contexts, both on the cell surface and within the cytoplasm, allowing protein localization to be imaged in live cells by epifluorescence and laser confocal microscopy.