Imaging protein-protein interactions inside living cells via interaction-dependent fluorophore ligation

We report a new method, Interaction-Dependent PRobe Incorporation Mediated by Enzymes, or ID-PRIME, for imaging protein-protein interactions (PPIs) inside living cells. ID-PRIME utilizes a mutant of Escherichia coli lipoic acid ligase, LplA(W37V), which can catalyze the covalent ligation of a coumarin fluorophore onto a peptide recognition sequence called LAP1. The affinity between the ligase and LAP1 is tuned such that, when each is fused to a protein partner of interest, LplA(W37V) labels LAP1 with coumarin only when the protein partners to which they are fused bring them together. Coumarin labeling in the absence of such interaction is low or undetectable. Characterization of ID-PRIME in living mammalian cells shows that multiple protein-protein interactions can be imaged (FRB-FKBP, Fos-Jun, and neuroligin-PSD-95), with as little as 10 min of coumarin treatment. The signal intensity and detection sensitivity are similar to those of the widely used fluorescent protein complementation technique (BiFC) for PPI detection, without the disadvantage of irreversible complex trapping. ID-PRIME provides a powerful and complementary approach to existing methods for visualization of PPIs in living cells with spatial and temporal resolution.     

Amide-based molecular knots as platforms for fluorescent switches

A series of amide-based molecular knots equipped selectively with fluorescent dansyl and/or pyrenesulfonyl moieties were synthesized from the readily available tris(allyloxy)knotane. UV/Vis absorption spectra, emission spectra, and the emission lifetimes of the fluorescent knotanes were investigated in chloroform at 298 K. The absorption spectra of the knotanes correspond to those of mixtures of their UV-active constituents. The fluorescence quantum yields and lifetimes of the dansyl and pyrenesulfonyl moieties are partly quenched by the knotane platform. In the KN(Da)(2)(Py) species, the fluorescent excited state of the dansyl units (lambda(max)=510 nm) lies at lower energy than the fluorescent excited state of the pyrenesulfonyl unit (lambda(max)=385 nm), the emission of which is accordingly quenched with sensitization of the dansyl fluorescence. In the KN(Ao)(2)(Da), KN(Ao)(Da)(2), and KN(Da)(3) species, the addition of acids causes the protonation of their dansyl units with a consequent decrease in the intensity of the dansyl band at 510 nm and appearance of the emission band of the protonated dansyl unit (lambda(max)=340 nm). Each dansyl unit of KN(Ao)(Da)(2) and KN(Da)(3) undergoes the independent protonation. In these incompletely protonated knotanes the fluorescence of the protonated dansyl units is partly quenched by nonprotonated ones. These processes can be quantitatively reversed upon addition of a base. In KN(Da)(2)(Py), an increase of the fluorescence of its pyrenesulfonyl group is observed when the dansyl groups are protonated. The results obtained show that the readily available and easily functionalizable amide-knotanes can be used as an interesting scaffold to obtain fluorescent switches.     

A selective fluorescent sensor for detecting lead in living cells

We present the synthesis, properties, and biological applications of Leadfluor-1 (LF1), a new water-soluble, turn-on fluorescent sensor that is capable of selectively imaging Pb2+ in aqueous solution and in living cells. LF1 combines a fluorescein chromophore and a pseudocrown receptor to provide good selectivity for Pb2+ over a range of biologically and environmentally relevant metal ions in aqueous solution, with sensitivity to parts per billion EPA limits for allowable lead in drinking water. In addition to these attributes, imaging experiments further show that LF1 is the first small-molecule reagent that can be delivered into living cells and report changes in intracellular Pb2+ levels.     

Diels-Alder cycloaddition for fluorophore targeting to specific proteins inside living cells

The inverse-electron-demand Diels-Alder cycloaddition between trans-cyclooctenes and tetrazines is biocompatible and exceptionally fast. We utilized this chemistry for site-specific fluorescence labeling of proteins on the cell surface and inside living mammalian cells by a two-step protocol. Escherichia coli lipoic acid ligase site-specifically ligates a trans-cyclooctene derivative onto a protein of interest in the first step, followed by chemoselective derivatization with a tetrazine-fluorophore conjugate in the second step. On the cell surface, this labeling was fluorogenic and highly sensitive. Inside the cell, we achieved specific labeling of cytoskeletal proteins with green and red fluorophores. By incorporating the Diels-Alder cycloaddition, we have broadened the panel of fluorophores that can be targeted by lipoic acid ligase.     

A selective reaction-based fluorescent probe for detecting cobalt in living cells

A reaction-based strategy exploiting cobalt-mediated oxidative C-O bond cleavage affords a selective turn-on fluorescent probe for paramagnetic Co(2+) in water and in living cells.     

A miniaturized arrayed assay format for detecting small molecule-protein interactions in cells

Background: Two complementary approaches to studying the cellular function of proteins involve alteration of function either by mutating protein-encoding genes or by binding a small molecule to the protein. A mutagen can generate millions of genetic mutations; correspondingly, split-pool synthesis can generate millions of unique ligands attached to individual beads. Genetic screening of mutations is relatively straightforward but, in contrast, split-pool synthesis presents a challenge to current methods of screening for compounds that alter protein function. The methods used to screen natural products are not feasible for large libraries composed of covalently immobilized compounds on synthesis beads. The sheer number of compounds synthesized by split-pool synthesis, and the small quantity of individual compound attached to each bead require assay miniaturization for efficient screening.Results: We present a miniaturized cell-based technique for the screening of ligands prepared by split-pool synthesis. Spatially defined droplets with uniform volumes of approximately 50–150 nanoliters (depending on well dimensions) are arrayed on plastic devices prepared using a combination of photolithography and polymer molding. Using this microtechnology, approximately 6,500 assays using either yeast cells or mammalian tissue culture can be performed within the dimensions of a standard 10 cm petri dish. We demonstrate that the biological effect of a small molecule prepared by split-pool synthesis can be detected in this format following its photorelease from a bead.Conclusions: The miniaturized format described here allows uniformly sized nanodroplets to be arrayed on plastic devices. The design is amenable to a large number of biological assays and the spatially arrayed format ensures uniform and controlled ligand concentrations and should facilitate automation of assays. The screening method presented here provides an efficient means of rapidly screening large numbers of ligands made by split-pool synthesis in both yeast and mammalian cells.     

Stereochemical control of calixarenes useful as rigid and conformationally diversiform platforms for molecular design

Stereochemical problems and related functions of calix[4]arenes, calix[6]arenes and their chiral derivatives have been reviewed. In p-tert-butylcalix[4]arene (1H₄) and its mono-, di-, tri-, and tetra-O-alkyl derivatives (1H₃R, 1H₂R₂,1HR₃, and 1R₄, respectively), 23 different homologues can exist (including 1H₄). We found that the OH group in the unmodified phenol unit is permeable through the calix[4]arene ring. Thus, several conformational isomers become equivalent after the ‘oxygen-through-the-annulus’ rotation of the OH group and the number of possible homologues is reduced to 13 (including 1H₃). We report in this paper the syntheses of all of these possible conformational isomers using a protection-deprotection method with a benzyl group and metal template effects. On the other hand, all possible chiral isomers that can be derived from calix[4]arene by modification of the OH groups have been systematically classified. Molecular asymmetry can be generated not only by different substituents but also by conformational isomerism. The numbers of chiral isomers are 17 for tetra-O-substituted calix[4]arenes, 9 for tri-O-substituted calix[4]arenes, 3 for di-O-substituted calix[4]arenes, and 0 for mono-O-substituted calix[4]arenes. Chiral calix[4]arenes can also be designed by the introduction of a substituent into the m-position of a phenol unit or by the use of a dissymmetric ‘stapling reaction’ in proximal phenol units. In p-tert-butylcalix[6]arene, the conformational behaviour is totally different from that in p-tert-butylcalix[4]arene. A large degree of conformational freedom remains in the framework, and both ‘oxygen-through-the-annulus rotation’ and ‘para-substituent-through-the-annulus rotation’ can take place. However, when metal cations are bound to calix[6]aryl esters, the conformation is changed to a cone type. Bridging and capping are powerful methods to immobilize the conformation of calix[6]arenes. In addition, definitive evidence for ring immobilization was obtained from the absence of racemization in the chiral calix[6]arene. A successful example of chiral recognition for α-amino acid derivatives was achieved by using chiral homooxacalix[3]arene which has ‘pseudo C₂ symmetry’. These examples indicate that calixarenes serve as rigid and conformationally diversiform platforms for the design of novel functional supramolecules.     

A ratiometric merocyanine-based fluorescent probe for detecting hydrazine in living cells and zebra fish

We developed a merocyanine-based fluorescent probe,NEPB,for tracing hydrazine(N_2 H_4) in a ratiometric manner with large Stokes shifts and long emission wavelength.The fluorescence color of probe NEPB changed from green to yellow upon addition of hydrazine.Probe NEPB displayed high selectivity and sensitivity to hydrazine in solution,and could ratiometrically monitor N_2 H_4 in living cells and zebrafish with low cytotoxicity.     

Glycogen phosphorylase as a molecular target for type 2 diabetes therapy

The regulation of the hepatic glucose output through glycogenolysis is an important target for type 2 diabetes therapy. Glycogenolysis is catalyzed in liver, muscle and brain by tissue specific isoforms of glycogen phosphorylase (GP). Because of its central role in glycogen metabolism, GP has been exploited as a model for structure-assisted design of potent inhibitors, which may be relevant to the control of blood glucose concentrations in type 2 diabetes. Several regulatory binding sites have been identified in GP, such as the catalytic, the allosteric, and the inhibitor binding sites. Protein crystallography has contributed significant structural information on the specificity and interactions that distinguish the binding sites, and also revealed a new unexpected binding site (new allosteric site). In this review, the kinetic, crystallographic binding, and physiological studies of a number of compounds, inhibitors of GP, are described, and the essential inhibitory and binding properties of specific compounds are analyzed in an effort to provide rationalizations for the affinities of these compounds and to exploit the molecular interactions that might give rise to a better inhibitor. These studies have given new insights into fundamental structural aspects of the enzyme enhancing our understanding of how the enzyme recognizes and specifically binds ligands, that could be of potential therapeutic value in the treatment of type 2 diabetes.     

Amphiphilic diarylethene as a photoswitchable probe for imaging living cells

This communication reports a unique example of water-soluble and fluorescent-switchable amphiphilic diarylethene. This compound performs stable vesicle aggregation in water and shows aggregation-dependent emission in its open form. The fluorescence can be effectively switched by alternating between UV and visible light irradiation. This compound thus can stain KB cells for switchable living cell imaging with excellent resistance to fatigue.     

A highly selective turn-on colorimetric, red fluorescent sensor for detecting mobile zinc in living cells

We describe ZRL1, a turn-on colorimetric and red fluorescent zinc ion sensor. The Zn(2+)-promoted ring opening of the rhodamine spirolactam ring in ZRL1 evokes a 220-fold fluorescence turn-on response. In aqueous media, ZRL1 turn-on luminescence is highly selective for Zn(2+) ions, with no significant response to other competitive cations, including Na(+), K(+), Ca(2+), Mg(2+), Mn(2+), Fe(2+), Co(2+), Ni(2+), Cu(2+), Cd(2+), or Hg(2+). In addition to these characteristics, preliminary results indicate that ZRL1 can be delivered to living cells and can be used to monitor changes in intracellular Zn(2+) levels.     

A turn-on fluorescent probe based on hydroxylamine oxidation for detecting ferric ion selectively in living cells

We have described a turn on fluorescent probe BOD-NHOH based on hydroxylamine oxidation for detecting intracellular ferric ions. The probe comprises a signal transducer of BODIPY dye and a Fe(3+)-response modulator of hydroxylamine. It is readily employed for assessing intracellular ferric ion levels, and confocal imaging is achieved successfully.     

Functionalized Fe3O4 nanoparticles for detecting zinc ions in living cells and their cytotoxicity

The zinc tank: A new fluoro-chromogenic chemosensor based on BODIPY-functionalized Fe(3)O(4) nanoparticles (1) has been prepared. Chemoprobe 1 exhibits high selectivity for Zn(2+) over other competing metal ions tested. Moreover, confocal microscopy experiments established that 1 can be used for detecting Zn(2+) levels in living cells (see figure).     

8-hydroxyquinoline derivatives as fluorescent sensors for magnesium in living cells

Despite the key role of magnesium in many fundamental biological processes, knowledge about its intracellular regulation is still scarce, due to the lack of appropriate detection methods. Here, we report the spectroscopic and photochemical characterization of two diaza-18-crown-6 hydroxyquinoline derivatives (DCHQ) and we propose their application in total Mg(2+) assessment and in confocal imaging as effective Mg(2+) indicators. DCHQ derivatives 1 and 2 bind Mg(2+) with much higher affinity than other available probes (K(d) = 44 and 73 microM, respectively) and show a strong fluorescence increase upon binding. Remarkably, fluorescence output is not significantly affected by other divalent cations, most importantly Ca(2+), or by pH changes within the physiological range. Evidence is provided on the use of fluorometric data to derive total cellular Mg(2+) content, which is consistent with atomic absorption data. Furthermore, we show that DCHQ compounds can be effectively employed to map intracellular ion distribution and movements in live cells by confocal microscopy. A clear staining pattern consistent with known affinities of Mg(2+) for biological ligands is shown; moreover, changes in the fluorescence signal could be tracked following stimuli known to modify intracellular Mg(2+) concentration. These findings suggest that DCHQ derivatives may serve as new tools for the study of Mg(2+) regulation, allowing sensitive and straightforward detection of both static and dynamic signals.     

Synthetic receptors as sensor coatings for molecules and living cells.

Non-covalent molecularly imprinted polymers are applied as sensitive coatings to planar waveguides and mass-sensitive devices for the selective detection of various groups of analytes in the gaseous and aqueous phases. Cavity imprinting in the bulk of the sensor material as well as surface imprinting techniques are used to enrich analytes ranging from sub-nanometres to micrometres in analyte size. The coated devices provide sensitivity to e.g. polycyclic aromatic hydrocarbons, xanthine derivatives, complex coffee samples and whole microorganisms.     

A novel,simple and sensitive ligand affinity capture method for detecting molecular interactions by MALDI mass spectrometry

A simple and sensitive ligand affinity capture method (LAC) was developed to detect biotinylated biomolecules bound to a biotin–avidin base by matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry (MALDI ToF MS). Glass slides covered with a metal film for MALDI MS applications were treated with amino‐silane and derivatized with biotin followed by binding of avidin. Washing buffers with high ionic strength increased the specificity of the subsequent binding of biotinylated biomolecules to the avidin layer. A combined thin layer‐dried droplet method using α‐cyano‐4‐hydroxycinnamic acid (CHCA) in acetone or ethyl acetate resulted in the most intense ions of biotinylated polymyxin B, whereas the matrix conditions did not influence the detection of angiotensin II. Addition of biotinylated biomolecules in the low femtomole to low picomole range resulted in sufficient ion intensity for detection by the LAC method. The LAC concept was extended by binding of biotinylated lipopolysaccharide to the biotin–avidin base followed by preferential capture and specific detection of the binding antagonist polymyxin B. Copyright © 2008 John Wiley & Sons, Ltd.