tetramethylrhodamine and 4-4-difluoro-4-bora-3a-4a-diaza-s-indacene

The structures, dynamics and energetics of the protonated, derivatized peptide DyeX-(Pro)(4)-Arg(+)-Trp, where "Dye" stands for the BODIPY analogue of tetramethylrhodamine and X is a (CH(2))(5) linker, have been investigated using a combination of modeling approaches in order to provide a numerical framework to the interpretation of fluorescence quenching data in the gas phase. Molecular dynamics (MD) calculations using the new generation AMOEBA force field were carried out using a representative set of conformations, at eight temperatures ranging from 150 to 500 K. Force field parameters were derived from ab initio calculations for the Dye. Strong electrostatic, polarization and dispersion interactions combine to shape this charged peptide. These effects arise in particular from the electric field generated by the charge of the protonated arginine and from several hydrogen bonds that can be established between the Dye linker and the terminal Trp. This conclusion is based on both the analysis of all structures generated in the MD simulations and on an energy decomposition analysis at classical and quantum mechanical levels. Structural analysis of the simulations at the different temperatures reveals that the relatively rigid polyproline segment allows for the Dye and Trp indole side chain to adopt stacking conformations favorable to electron transfer, yielding support to a model in which it is electron transfer from tryptophan to the dye that drives fluorescence quenching.

Topics: Amino Acid Sequence; Boron Compounds; Electron Transport; Hydrogen Bonding; Molecular Dynamics Simulation; Peptides; Quantum Theory; Rhodamines; Static Electricity; Temperature; Thermodynamics

Metabolic cytometry is a form of chemical cytometry wherein metabolic cascades are monitored in single cells. We report the first example of metabolic cytometry where two different metabolic pathways are simultaneously monitored. Glycolipid catabolism in primary rat cerebella neurons was probed by incubation with tetramethylrhodamine-labeled GM1 (GM1-TMR). Simultaneously, both catabolism and anabolism were probed by coincubation with BODIPY-FL labeled LacCer (LacCer-BODIPY-FL). In a metabolic cytometry experiment, single cells were incubated with substrate, washed, aspirated into a capillary, and lysed. The components were separated by capillary electrophoresis equipped with a two-spectral channel laser-induced fluorescence detector. One channel monitored fluorescence generated by the metabolic products produced from GM1-TMR and the other monitored the metabolic products produced from LacCer-BODIPY-FL. The metabolic products were identified by comparison with the mobility of a set of standards. The detection system produced at least 6 orders of magnitude dynamic range in each spectral channel with negligible spectral crosstalk. Detection limits were 1 zmol for BODIPY-FL and 500 ymol for tetramethylrhodamine standard solutions.

Topics: Animals; Boron Compounds; Brain; Cells, Cultured; Electrophoresis, Capillary; Equipment Design; Fluorescence; Fluorescent Dyes; Glycosphingolipids; Lactosylceramides; Limit of Detection; Metabolic Networks and Pathways; Microscopy, Fluorescence; Neurons; Rats; Rhodamines

Protein and lipid kinases are two important classes of biomedically relevant enzymes. The expression and activity of many kinases are known to be dysregulated in a variety of diseases, and proteomic tools that can assess the presence and activity of these enzymes are likely to be useful for their evaluation. Because many of the mechanisms by which protein kinases can become unregulated involve post-translational modifications or changes in protein localization, they can only be detected by examining protein activity, sometimes within the context of the living cell. Wortmannin is a steroid-derived fungal metabolite that covalently inhibits both protein and lipid kinases. Here we describe the synthesis of three wortmannin derivatives, biotin-wortmannin, BODIPY-wortmannin, and tetramethylrhodamine-wortmannin. We demonstrate that these reagents exhibit reactivity similarly as wortmannin and react with members of the phosphatidylinositol 3-kinase and PI3-kinase related kinase families in cellular lysates. Moreover, in some cases these reagents can differentiate between the active and inactive forms of the enzyme, indicating that they are activity-based probes. The reagents also exhibit complementary properties. The biotin-wortmannin reagent is effective in the isolation of labeled proteins; all three can be used for protein labeling, and BODIPY-wortmannin is cell-permeable and can be used to label proteins within cells.

Topics: Androstadienes; Biochemistry; Biotin; Boron Compounds; Cell Line; Enzyme Inhibitors; Fluorescent Dyes; Glutathione Transferase; HeLa Cells; Humans; Inhibitory Concentration 50; Lipids; Models, Chemical; Phosphatidylinositol 3-Kinases; Phosphorylation; Protein Binding; Protein Processing, Post-Translational; Rhodamines; Time Factors; Transfection; Wortmannin

Here we describe a high-throughput, quantitative method for the isolation of enzymes with novel substrate specificities from large libraries of protein variants. Protein variants are displayed on the surface of microorganisms and incubated with a synthetic substrate consisting of (1) a fluorescent dye (2) a positively charged moiety (3) the target scissile bond, and (4) a fluorescence resonance energy transfer (FRET) quenching partner. Enzymatic cleavage of the scissile bond results in release of the FRET quenching partner while the fluorescent product is retained on the cell surface, allowing isolation of catalytically active clones by fluorescence-activated cell sorting (FACS). Using a synthetic substrate with these characteristics, we enriched Escherichia coli expressing the serine protease OmpT from cells expressing an inactive OmpT variant by over 5,000-fold in a single round. Screening a library of 6 x 10(5) random OmpT variants by FACS using a FRET peptide substrate with a nonpreferred Arg-Val cleavage sequence resulted in the isolation of variant proteases with catalytic activities enhanced by as much as 60-fold. This approach represents a potentially widely applicable method for high-throughput screening of large libraries on the basis of catalytic turnover.

Topics: Boron Compounds; Catalysis; Cloning, Molecular; Energy Transfer; Enzymes; Escherichia coli; Flow Cytometry; Fluorescence; Fluorescent Dyes; Genetic Variation; Kinetics; Mutation; Peptide Library; Rhodamines; Serine Endopeptidases; Substrate Specificity

A strategy was developed to label specified sites in living cells with a wide selection of fluorescent or other probes and applied to study pH regulation in Golgi. cDNA transfection was used to target a single-chain antibody to a specified site such as an organelle lumen. The targeted antibody functioned as a high affinity receptor to trap cell-permeable hapten-fluorophore conjugates. Synthesized conjugates of a hapten (4-ethoxymethylene-2-phenyl-2-oxazolin-5-one, phOx) and fluorescent probes (Bodipy Fl, tetramethylrhodamine, fluorescein) were bound with high affinity (approximately 5 nM) and specific localization to the single-chain antibody expressed in the endoplasmic reticulum, Golgi, and plasma membrane of living Chinese hamster ovary cells. Using the pH-sensitive phOx-fluorescein conjugate and ratio imaging microscopy, pH was measured in the lumen of Golgi (pH 6.25 +/- 0.06). Measurements of pH-dependent vacuolar H+/ATPase pump activity and H+ leak in Golgi provided direct evidence that resting Golgi pH is determined by balanced leak-pump kinetics rather than the inability of the H+/ATPase to pump against an electrochemical gradient. Like expression of the green fluorescent protein, the receptor-mediated fluorophore targeting approach permits specific intracellular fluorescence labeling. A significant advantage of the new approach is the ability to target chemical probes with custom-designed spectral and indicator properties.

Topics: Adjuvants, Immunologic; Affinity Labels; Animals; Boron Compounds; CHO Cells; Cricetinae; Fluorescein; Fluorescent Dyes; Golgi Apparatus; Haptens; Hydrogen-Ion Concentration; Immunoglobulin Fragments; Models, Chemical; Oxazolone; Proton-Translocating ATPases; Rhodamines; Thermodynamics