title: Multiexcitation Fluorogenic Labeling of Surface, Intracellular, and Total Protein Pools in Living Cells.
abstract: Malachite green (MG) is a fluorogenic dye that shows fluorescence enhancement upon binding to its engineered cognate protein, a fluorogen activating protein (FAP). Energy transfer donors such as cyanine and rhodamine dyes have been conjugated with MG to modify the spectral properties of the fluorescent complexes, where the donor dyes transfer energy through Förster resonance energy transfer to the MG complex resulting in binding-conditional fluorescence emission in the far-red region. In this article, we use a violet-excitable dye as a donor to sensitize the far-red emission of the MG-FAP complex. Two blue emitting fluorescent coumarin dyes were coupled to MG and evaluated for energy transfer to the MG-FAP complex via its secondary excitation band. 6,8-Difluoro-7-hydroxycoumarin-3-carboxylic acid (Pacific blue, PB) showed the most efficient energy transfer and maximum brightness in the far-red region upon violet (405 nm) excitation. These blue-red (BluR) tandem dyes are spectrally varied from other tandem dyes and are able to produce fluorescence images of the MG-FAP complex with a large Stokes shift (>250 nm). These dyes are cell-permeable and are used to label intracellular proteins. Used together with a cell-impermeable hexa-Cy3-MG (HCM) dye that labels extracellular proteins, we are able to visualize extracellular, intracellular, and total pools of cellular protein using one fluorogenic tag that combines with distinct dyes to effect different spectral characteristics.
authors.affiliation: Department of Chemistry, ‡Department of Biological Sciences, and §Molecular Biosensors and Imaging Center, Carnegie Mellon University , 4400 Fifth Avenue, Pittsburgh 15213, Pennsylvania, United States.
authors.forename: Matharishwan
authors.initials: M
authors.lastname: Naganbabu
authors.affiliation: Department of Chemistry, ‡Department of Biological Sciences, and §Molecular Biosensors and Imaging Center, Carnegie Mellon University , 4400 Fifth Avenue, Pittsburgh 15213, Pennsylvania, United States.
authors.forename: Lydia A
authors.initials: LA
authors.lastname: Perkins
authors.affiliation: Department of Chemistry, ‡Department of Biological Sciences, and §Molecular Biosensors and Imaging Center, Carnegie Mellon University , 4400 Fifth Avenue, Pittsburgh 15213, Pennsylvania, United States.
authors.forename: Yi
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Department of Chemistry, ‡Department of Biological Sciences, and §Molecular Biosensors and Imaging Center, Carnegie Mellon University , 4400 Fifth Avenue, Pittsburgh 15213, Pennsylvania, United States.
authors.forename: Jeffery
authors.initials: J
authors.lastname: Kurish
authors.affiliation: Department of Chemistry, ‡Department of Biological Sciences, and §Molecular Biosensors and Imaging Center, Carnegie Mellon University , 4400 Fifth Avenue, Pittsburgh 15213, Pennsylvania, United States.
authors.forename: Brigitte F
authors.initials: BF
authors.lastname: Schmidt
authors.affiliation: Department of Chemistry, ‡Department of Biological Sciences, and §Molecular Biosensors and Imaging Center, Carnegie Mellon University , 4400 Fifth Avenue, Pittsburgh 15213, Pennsylvania, United States.
authors.forename: Marcel P
authors.initials: MP
authors.lastname: Bruchez
mesh_terms: D002470:Cell Survival; D005456:Fluorescent Dyes / Q000737:chemistry*; D057809:HEK293 Cells; D006801:Humans; D042541:Intracellular Space / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry*; D012394:Rosaniline Dyes / Q000737:chemistry*; D013499:Surface Properties
chemical_list: D005456:Fluorescent Dyes; D011506:Proteins; D012394:Rosaniline Dyes; C005095:malachite green