title: Fluorogenic affinity label for the facile, rapid imaging of proteins in live cells.
abstract: Haloalkane dehalogenase (HD) catalyzes the hydrolysis of haloalkanes via a covalent enzyme-substrate intermediate. Fusing a target protein to an HD variant that cannot hydrolyze the intermediate enables labeling of the target protein with a haloalkane in cellulo. The utility of extant probes is hampered, however, by background fluorescence as well as limited membrane permeability. Here, we report on the synthesis and use of a fluorogenic affinity label that, after unmasking by an intracellular esterase, labels an HD variant in cellulo. Labeling is rapid and specific, as expected from the reliance upon enzymic catalysts and the high membrane permeance of the probe both before and after unmasking. Most notably, even high concentrations of the fluorogenic affinity label cause minimal background fluorescence without a need to wash the cells. We envision that such fluorogenic affinity labels, which enlist catalysis by two cellular enzymes, will find utility in pulse-chase experiments, high-content screening, and numerous other protocols.
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, Madison, WI 53706, USA.
authors.forename: Rex W
authors.initials: RW
authors.lastname: Watkins
authors.forename: Luke D
authors.initials: LD
authors.lastname: Lavis
authors.forename: Vanessa M
authors.initials: VM
authors.lastname: Kung
authors.forename: Georgyi V
authors.initials: GV
authors.lastname: Los
authors.forename: Ronald T
authors.initials: RT
authors.lastname: Raines
mesh_terms: D000345:Affinity Labels / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D002470:Cell Survival; D005456:Fluorescent Dyes / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism*; D006867:Hydrolases / Q000378:metabolism*; D057927:Hydrophobic and Hydrophilic Interactions; D013997:Time Factors
chemical_list: D000345:Affinity Labels; D005456:Fluorescent Dyes; D006867:Hydrolases; C046137:haloalkane dehalogenase