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Sample records for fluorescent ligand 4-acridinol-1-sulphonic

  1. Fluorescent Ligands for Adenosine Receptors

    PubMed Central

    Kozma, Eszter; Jayasekara, P Suresh; Squarcialupi, Lucia; Paoletta, Silvia; Moro, Stefano; Federico, Stephanie; Spalluto, Giampiero; Jacobson, Kenneth A.

    2012-01-01

    Interest is increasing in developing fluorescent ligands for characterization of adenosine receptors (ARs), which hold a promise of usefulness in the drug discovery process. The size of a strategically labeled AR ligand can be greatly increased after the attachment of a fluorophore. The choice of dye moiety (e.g. Alexa Fluor 488), attachment point and linker length can alter the selectivity and potency of the parent molecule. Fluorescent derivatives of adenosine agonists and antagonists (e.g. XAC and other heterocyclic antagonist scaffolds) have been synthesized and characterized pharmacologically. Some are useful AR probes for flow cytometry, fluorescence correlation spectroscopy, fluorescence microscopy, fluorescence polarization, fluorescence resonance energy transfer, and scanning confocal microscopy. Thus, the approach of fluorescent labeled GPCR ligands, including those for ARs, is a growing dynamic research field. PMID:23200243

  2. Bioconjugation Methods for Coupling Targeting Ligands with Fluorescent Dyes.

    PubMed

    Ling, Xiaoxi

    2016-01-01

    Targeted molecular imaging probes are essential tools for visualization of specific molecular processes in cells and living systems. Among these, targeted fluorescent probes are widely used due to the high sensitivity and resolution of fluorescence imaging. The conventional strategy for developing targeted fluorescent probes is to couple targeting ligands with fluorescent dyes by covalent bond via bioconjugation. Here, we describe several commonly used bioconjugation methods, from traditional amide and thiol coupling, to metal-catalyzed coupling reaction and catalyst free cycloaddition. PMID:27283413

  3. Fluorescent ligand for human progesterone receptor imaging in live cells.

    PubMed

    Weinstain, Roy; Kanter, Joan; Friedman, Beth; Ellies, Lesley G; Baker, Michael E; Tsien, Roger Y

    2013-05-15

    We employed molecular modeling to design and then synthesize fluorescent ligands for the human progesterone receptor. Boron dipyrromethene (BODIPY) or tetramethylrhodamine were conjugated to the progesterone receptor antagonist RU486 (Mifepristone) through an extended hydrophilic linker. The fluorescent ligands demonstrated comparable bioactivity to the parent antagonist in live cells and triggered nuclear translocation of the receptor in a specific manner. The BODIPY labeled ligand was applied to investigate the dependency of progesterone receptor nuclear translocation on partner proteins and to show that functional heat shock protein 90 but not immunophilin FKBP52 activity is essential. A tissue distribution study indicated that the fluorescent ligand preferentially accumulates in tissues that express high levels of the receptor in vivo. The design and properties of the BODIPY-labeled RU486 make it a potential candidate for in vivo imaging of PR by positron emission tomography through incorporation of (18)F into the BODIPY core. PMID:23600997

  4. Fluorescent and Lanthanide Labeling for Ligand Screens, Assays, and Imaging

    PubMed Central

    Josan, Jatinder S.; De Silva, Channa R.; Yoo, Byunghee; Lynch, Ronald M.; Pagel, Mark D.; Vagner, Josef; Hruby, Victor J.

    2012-01-01

    The use of fluorescent (or luminescent) and metal contrast agents in high-throughput screens, in vitro assays, and molecular imaging procedures has rapidly expanded in recent years. Here we describe the development and utility of high-affinity ligands for cancer theranostics and other in vitro screening studies. In this context, we also illustrate the syntheses and use of heteromultivalent ligands as targeted imaging agents. PMID:21318902

  5. Analysis of protein-ligand interactions by fluorescence polarization

    PubMed Central

    Rossi, Ana M.; Taylor, Colin W.

    2011-01-01

    Quantification of the associations between biomolecules is required both to predict and understand the interactions that underpin all biological activity. Fluorescence polarization (FP) provides a non-disruptive means of measuring the association of a fluorescent ligand with a larger molecule. We describe an FP assay in which binding of fluorescein-labelled inositol 1,4,5-trisphosphate (IP3) to N-terminal fragments of IP3 receptors can be characterised at different temperatures and in competition with other ligands. The assay allows the standard Gibbs free energy (ΔG°), enthalpy (ΔH°) and entropy (ΔS°) changes of ligand binding to be determined. The method is applicable to any purified ligand-binding site for which an appropriate fluorescent ligand is available. FP can be used to measure low-affinity interactions in real-time without use of radioactive materials, it is non-destructive, and with appropriate care it can resolve ΔH° and ΔS°. The first part of the protocol, protein preparation, may take several weeks, while the FP measurements, once they have been optimised, would normally take 1-6 h. PMID:21372817

  6. Portraying G Protein-Coupled Receptors with Fluorescent Ligands

    PubMed Central

    2015-01-01

    The thermodynamics of ligand–receptor interactions at the surface of living cells represents a fundamental aspect of G protein-coupled receptor (GPCR) biology; thus, its detailed elucidation constitutes a challenge for modern pharmacology. Interestingly, fluorescent ligands have been developed for a variety of GPCRs in order to monitor ligand–receptor binding in living cells. Accordingly, new methodological strategies derived from noninvasive fluorescence-based approaches, especially fluorescence resonance energy transfer (FRET), have been successfully developed to characterize ligand–receptor interactions. Importantly, these technologies are supplanting more hazardous and expensive radioactive binding assays. In addition, FRET-based tools have also become extremely powerful approaches for visualizing receptor–receptor interactions (i.e., GPCR oligomerization) in living cells. Thus, by means of the synthesis of compatible fluorescent ligands these novel techniques can be implemented to demonstrate the existence of GPCR oligomerization not only in heterologous systems but also in native tissues. Finally, there is no doubt that these methodologies would also be relevant in drug discovery in order to develop new high-throughput screening approaches or to identify new therapeutic targets. Overall, herein, we provide a thorough assessment of all technical and biological aspects, including strengths and weaknesses, of these fluorescence-based methodologies when applied to the study of GPCR biology at the plasma membrane of living cells. PMID:25010291

  7. Ligand screening using fluorescence thermal shift analysis (FTS).

    PubMed

    Luan, Chi-Hao; Light, Samuel H; Dunne, Sara F; Anderson, Wayne F

    2014-01-01

    The fluorescence thermal shift (FTS) method is a biophysical technique that can improve productivity in a structural genomics pipeline and provide a fast and easy platform for identifying ligands in protein function or drug discovery screening. The technique has gained widespread popularity in recent years due to its broad-scale applicability, throughput, and functional relevance. FTS is based on the principle that a protein unfolds at a critical temperature that depends upon its intrinsic stability. A probe that will fluoresce when bound to hydrophobic surfaces is used to monitor protein unfolding as temperature is increased. In this manner, conditions or small molecules that affect the thermal stability of a protein can be identified. Herein, principles, protocols, data analysis, and special considerations of FTS screening as performed for the Center for Structural Genomics of Infectious Diseases (CSGID) pipeline are described in detail. The CSGID FTS screen is designed as a high-throughput 384-well assay to be performed on a robotic platform; however, all protocols can be adapted to a 96-well format that can be assembled manually. Data analysis can be performed using a simple curve fitting of the fluorescent signal using a Boltzmann or double Boltzmann equation. A case study of 100 proteins screened against Emerald Biosystem's ADDit™ library is included as discussion. PMID:24590724

  8. A fluorescent approach for identifying P2X1 ligands

    PubMed Central

    Ruepp, Marc-David; Brozik, James A.; de Esch, Iwan J.P.; Farndale, Richard W.; Murrell-Lagnado, Ruth D.; Thompson, Andrew J.

    2015-01-01

    There are no commercially available, small, receptor-specific P2X1 ligands. There are several synthetic derivatives of the natural agonist ATP and some structurally-complex antagonists including compounds such as PPADS, NTP-ATP, suramin and its derivatives (e.g. NF279, NF449). NF449 is the most potent and selective ligand, but potencies of many others are not particularly high and they can also act at other P2X, P2Y and non-purinergic receptors. While there is clearly scope for further work on P2X1 receptor pharmacology, screening can be difficult owing to rapid receptor desensitisation. To reduce desensitisation substitutions can be made within the N-terminus of the P2X1 receptor, but these could also affect ligand properties. An alternative is the use of fluorescent voltage-sensitive dyes that respond to membrane potential changes resulting from channel opening. Here we utilised this approach in conjunction with fragment-based drug-discovery. Using a single concentration (300 μM) we identified 46 novel leads from a library of 1443 fragments (hit rate = 3.2%). These hits were independently validated by measuring concentration-dependence with the same voltage-sensitive dye, and by visualising the competition of hits with an Alexa-647-ATP fluorophore using confocal microscopy; confocal yielded kon (1.142 × 106 M−1 s−1) and koff (0.136 s−1) for Alexa-647-ATP (Kd = 119 nM). The identified hit fragments had promising structural diversity. In summary, the measurement of functional responses using voltage-sensitive dyes was flexible and cost-effective because labelled competitors were not needed, effects were independent of a specific binding site, and both agonist and antagonist actions were probed in a single assay. The method is widely applicable and could be applied to all P2X family members, as well as other voltage-gated and ligand-gated ion channels. This article is part of the Special Issue entitled ‘Fluorescent Tools in Neuropharmacology

  9. Probing Ternary Complex Equilibria of Crown Ether Ligands by Time-Resolved Fluorescence Spectroscopy

    PubMed Central

    2015-01-01

    Ternary complex formation with solvent molecules and other adventitious ligands may compromise the performance of metal-ion-selective fluorescent probes. As Ca(II) can accommodate more than 6 donors in the first coordination sphere, commonly used crown ether ligands are prone to ternary complex formation with this cation. The steric strain imposed by auxiliary ligands, however, may result in an ensemble of rapidly equilibrating coordination species with varying degrees of interaction between the cation and the specific donor atoms mediating the fluorescence response, thus diminishing the change in fluorescence properties upon Ca(II) binding. To explore the influence of ligand architecture on these equilibria, we tethered two structurally distinct aza-15-crown-5 ligands to pyrazoline fluorophores as reporters. Due to ultrafast photoinduced electron-transfer (PET) quenching of the fluorophore by the ligand moiety, the fluorescence decay profile directly reflects the species composition in the ground state. By adjusting the PET driving force through electronic tuning of the pyrazoline fluorophores, we were able to differentiate between species with only subtle variations in PET donor abilities. Concluding from a global analysis of the corresponding fluorescence decay profiles, the coordination species composition was indeed strongly dependent on the ligand architecture. Altogether, the combination of time-resolved fluorescence spectroscopy with selective tuning of the PET driving force represents an effective analytical tool to study dynamic coordination equilibria and thus to optimize ligand architectures for the design of high-contrast cation-responsive fluorescence switches. PMID:25313708

  10. Novel Chalcone-Based Fluorescent Human Histamine H3 Receptor Ligands as Pharmacological Tools

    PubMed Central

    Tomasch, Miriam; Schwed, J. Stephan; Weizel, Lilia; Stark, Holger

    2012-01-01

    Novel fluorescent chalcone-based ligands at human histamine H3 receptors (hH3R) have been designed, synthesized, and characterized. Compounds described are non-imidazole analogs of ciproxifan with a tetralone motif. Tetralones as chemical precursors and related fluorescent chalcones exhibit affinities at hH3R in the same concentration range like the reference antagonist ciproxifan (hH3R pKi value of 7.2). Fluorescence characterization of our novel ligands shows emission maxima about 570 nm for yellow fluorescent chalcones and ≥600 nm for the red fluorescent derivatives. Interferences to cellular autofluorescence could be excluded. All synthesized chalcone compounds could be used to visualize hH3R proteins in stably transfected HEK-293 cells using confocal laser scanning fluorescence microscopy. These novel fluorescent ligands possess high potential to be used as pharmacological tools for hH3R visualization in different tissues. PMID:22470321

  11. Development of a quantitative fluorescence-based ligand-binding assay.

    PubMed

    Breen, Conor J; Raverdeau, Mathilde; Voorheis, H Paul

    2016-01-01

    A major goal of biology is to develop a quantitative ligand-binding assay that does not involve the use of radioactivity. Existing fluorescence-based assays have a serious drawback due to fluorescence quenching that accompanies the binding of fluorescently-labeled ligands to their receptors. This limitation of existing fluorescence-based assays prevents the number of cellular receptors under investigation from being accurately measured. We have developed a method where FITC-labeled proteins bound to a cell surface are proteolyzed extensively to eliminate fluorescence quenching and then the fluorescence of the resulting sample is compared to that of a known concentration of the proteolyzed FITC-protein employed. This step enables the number of cellular receptors to be measured quantitatively. We expect that this method will provide researchers with a viable alternative to the use of radioactivity in ligand binding assays. PMID:27161290

  12. Development of a quantitative fluorescence-based ligand-binding assay

    PubMed Central

    Breen, Conor J.; Raverdeau, Mathilde; Voorheis, H. Paul

    2016-01-01

    A major goal of biology is to develop a quantitative ligand-binding assay that does not involve the use of radioactivity. Existing fluorescence-based assays have a serious drawback due to fluorescence quenching that accompanies the binding of fluorescently-labeled ligands to their receptors. This limitation of existing fluorescence-based assays prevents the number of cellular receptors under investigation from being accurately measured. We have developed a method where FITC-labeled proteins bound to a cell surface are proteolyzed extensively to eliminate fluorescence quenching and then the fluorescence of the resulting sample is compared to that of a known concentration of the proteolyzed FITC-protein employed. This step enables the number of cellular receptors to be measured quantitatively. We expect that this method will provide researchers with a viable alternative to the use of radioactivity in ligand binding assays. PMID:27161290

  13. Boron difluorides with formazanate ligands: redox-switchable fluorescent dyes with large stokes shifts.

    PubMed

    Chang, M-C; Chantzis, A; Jacquemin, D; Otten, E

    2016-06-21

    The synthesis of a series of (formazanate)boron difluorides and their 1-electron reduction products is described. The neutral compounds are fluorescent with large Stokes shifts. DFT calculations suggest that a large structural reorganization accompanies photoexictation and accounts for the large Stokes shift. Reduction of the neutral boron difluorides occurs at the ligand and generates the corresponding radical anions. These complexes are non-fluorescent, allowing switching of the emission by changing the ligand oxidation state. PMID:27188989

  14. How to Illustrate Ligand-Protein Binding in a Class Experiment: An Elementary Fluorescent Assay.

    ERIC Educational Resources Information Center

    Marty, Alain; And Others

    1986-01-01

    Describes an experiment (taking approximately five hours) which illustrates the binding of a small molecule to a protein. By using an appropriate fluorescent ligand and a given protein, the fluorescent probe technique is applied to measure the number of bonding sites, and number of site classes, and their association constants. (JN)

  15. Fluorescent Approaches for Understanding Interactions of Ligands with G Protein Coupled Receptors

    PubMed Central

    Sridharan, Rajashri; Zuber, Jeffrey; Connelly, Sara M.; Mathew, Elizabeth; Dumont, Mark E.

    2014-01-01

    G Protein Coupled Receptors (GPCRs) are responsible for a wide variety of signaling responses in diverse cell types. Despite major advances in the determination of structures of this class of receptors, the underlying mechanisms by which binding of different types of ligands specifically elicits particular signaling responses remains unclear. The use of fluorescence spectroscopy can provide important information about the process of ligand binding and ligand dependent conformational changes in receptors, especially kinetic aspects of these processes, that can be difficult to extract from x-ray structures. We present an overview of the extensive array of fluorescent ligands that have been used in studies of GPCRs and describe spectroscopic approaches for assaying binding and probing the environment of receptor-bound ligands with particular attention to examples involving yeast pheromone receptors. In addition, we discuss the use of fluorescence spectroscopy for detecting and characterizing conformational changes in receptors induced by the binding of ligands. Such studies have provided strong evidence for diversity of receptor conformations elicited by different ligands, consistent with the idea that GPCRs are not simple on and off switches. This diversity of states constitutes an underlying mechanistic basis for biased agonism, the observation that different stimuli can produce different responses from a single receptor. It is likely that continued technical advances will allow fluorescence spectroscopy to play an important role in continued probing of structural transitions in GPCRs. PMID:24055822

  16. Ligand Assisted Stabilization of Fluorescence Nanoparticles; an Insight on the Fluorescence Characteristics, Dispersion Stability and DNA Loading Efficiency of Nanoparticles.

    PubMed

    Rhouati, Amina; Hayat, Akhtar; Mishra, Rupesh K; Bueno, Diana; Shahid, Shakir Ahmad; Muñoz, Roberto; Marty, Jean Louis

    2016-07-01

    This work reports on the ligand assisted stabilization of Fluospheres® carboxylate modified nanoparticles (FCMNPs), and subsequently investigation on the DNA loading capacity and fluorescence response of the modified particles. The designed fluorescence bioconjugate was characterized with enhanced fluorescence characteristics, good stability and large surface area with high DNA loading efficiency. For comparison purpose, bovine serum albumin (BSA) and polyethylene glycol (PEG) with three different length strands were used as cross linkers to modify the particles, and their DNA loading capacity and fluorescence characteristics were investigated. By comparing the performance of the particles, we found that the most improved fluorescence characteristics, enhanced DNA loading and high dispersion stability were obtained, when employing PEG of long spacer arm length. The designed fluorescence bioconjugate was observed to maintain all its characteristics under varying pH over an extended period of time. These types of bioconjugates are in great demand for fluorescence imaging and in vivo fluorescence biomedical application, especially when most of the as synthesized fluorescence particles cannot withstand to varying in vivo physiological conditions with decreases in fluorescence response and DNA loading efficiency. PMID:27209005

  17. Intracavitary ligand distribution in tear lipocalin by site-directed tryptophan fluorescence.

    PubMed

    Gasymov, Oktay K; Abduragimov, Adil R; Glasgow, Ben J

    2009-08-01

    Site-directed tryptophan fluorescence has been successfully used to determine the solution structure of tear lipocalin. Here, the technique is extended to measure the binding energy landscape. Single Trp mutants of tear lipocalin are bound to the native ligand and an analogue tagged with a quencher group to both populate and discriminate the excited protein states. Steady-state and time-resolved fluorescence quenching data reveal the intracavitary state of the ligand. The static components of fluorescence quenching identify the residues where nonfluorescence complexes form. An asymmetric distribution of the ligand within the cavity reflects the complex energy landscape of the excited protein states. These findings suggest that the excited protein states are not unique but consist of many substates. The roughness of the binding energy landscape is about 2.5kBT. The excited protein states originate primarily from conformational selections of loops AB and GH, a portal region. In contrast to static quenching, the dynamic components of fluorescence quenching by the ligand are relevant to both local side chain and ligand dynamics. Apparent bimolecular rate constants for collisional quenching of Trp by the nitroxide moiety are approximately 1 / 5 x 10(12) M(-1) s(-1). Estimations made for effective ligand concentrations establish actual rate constants on the order of 12 x 10(9) M(-1) s(-1). Prior to exit from the cavity of the protein, ligands explore binding sites in nanoseconds. Although microsecond fluctuations are rate-limiting processes in ligand binding for many proteins, accompanying nanosecond motion may be necessary for propagation of ligand binding. PMID:19586017

  18. Quantitative Determination of DNA-Ligand Binding Using Fluorescence Spectroscopy

    ERIC Educational Resources Information Center

    Healy, Eamonn F.

    2007-01-01

    The effective use of fluorescence spectroscopy for determining the binding of the intercalcating agent crhidium bromide to DNA is being described. The analysis used simple measurement techniques and hence can be easily adopted by the students for a better understanding.

  19. Fluorogenic Enhancement of an in Vitro-Selected Peptide Ligand by Replacement of a Fluorescent Group.

    PubMed

    Wang, Wei; Zhu, Liping; Hirano, Yoshinori; Kariminavargani, Marziyeh; Tada, Seiichi; Zhang, Guanxin; Uzawa, Takanori; Zhang, Deqing; Hirose, Takuji; Taiji, Makoto; Ito, Yoshihiro

    2016-08-16

    To prepare a fluorogenic peptide ligand which binds to an arbitrary target, we previously succeeded in seeking a fluorogenic ligand to calmodulin using in vitro selection. In this study the environment-sensitive fluorescent group in the selected peptide ligand was replaced with other fluorescent groups to find the possibility to increase the fluorogenic activity. Surface plasmon resonance measurement exhibited that the binding affinity was held even after the replacement. However, the replacement significantly affected the fluorogenic activity. It depended on the kind of incorporated fluorophors and linker length. As a result, the incorporation of 4-N,N-dimethylamino-1,8-naphthalimide enhanced the fluorescence intensity over 100-fold in the presence of target calcium-bound calmodulin. This study demonstrated that the functionality of in vitro selected peptide can be tuned with keeping the binding affinity. PMID:27459509

  20. A Novel Cobalt-Sensitive Fluorescent Chemosensor Based on Ligand Capped CdS Quantum Dots.

    PubMed

    Faridbod, Farnoush; Jamali, Abbas; Ganjali, Mohammad Reza; Hosseini, Morteza; Norouzi, Parviz

    2015-05-01

    In this work, a ligand capped CdS QDs was synthesized, characterized and its fluorescence behavior was studied. The surface of the CdS QDs was modified using N-(3-methyl-2-(thiophene-2-carboxamido) phenyl) thiophene-2-carboxamide. The immobilized ligand on the surface of the CdS QDs can interact by cationic species due to the existence of donating atoms in its structures. Thus, effect of some metal cations on the fluorescent intensity of the ligand capped CdS QDs were studied. It was found that fluorescence intensity of the modified CdS QDs quenched selectively by addition of Co(II) ion in comparison with other cations tested. The ligand capped CdS QDs can be used as a fluorescent bulk chemosensor for detection of Co(II) ions. The fluorescent quenching is linear in the range of 1.0 × 10(-5) to 1.5 × 10(-4) mol L(-1) of Co(II) ions. The limit of detection was obtained 8.3 × 10(-7) mol L(-1). The nanosensor exhibits high selectivity toward Co(II) ions in comparison with common metal ions. PMID:25804832

  1. Characterization and Evaluation of Two Novel Fluorescent Sigma-2 Receptor Ligands as Proliferation Probes

    PubMed Central

    Zeng, Chenbo; Vangveravong, Suwanna; Jones, Lynne A.; Hyrc, Krzysztof; Chang, Katherine C.; Xu, Jinbin; Rothfuss, Justin M.; Goldberg, Mark P.; Hotchkiss, Richard S.; Mach, Robert H.

    2015-01-01

    We synthesized and characterized two novel fluorescent sigma-2 receptor selective ligands, SW120 and SW116, and evaluated these ligands as potential probes for imaging cell proliferation. Both ligands are highly selective for sigma-2 receptors versus sigma-1 receptors. SW120 and SW116 were internalized into MDA-MB-435 cells, and 50% of the maximum fluorescent intensity was reached in 11 and 24 minutes, respectively. In vitro studies showed that 50% of SW120 or SW116 washed out of cells in 1 hour. The internalization of SW120 was reduced ≈30% by phenylarsine oxide, an inhibitor of endocytosis, suggesting that sigma-2 ligands are internalized, in part, by an endocytotic pathway. Subcellular localization studies using confocal and two-photon microscopy showed that SW120 and SW116 partially colocalized with fluorescent markers of mitochondria, endoplasmic reticulum, lysosomes, and the plasma membrane, suggesting that sigma-2 receptors localized to the cytoplasmic organelles and plasma membrane. SW120 did not colocalize with the nuclear dye 4′,6-diamidino-2-phenylindole. In vivo studies showed that the uptake of SW120 in solid tumors and peripheral blood mononuclear cells of mice positively correlated with the expression level of the cell proliferation marker Ki-67, suggesting that sigma-2 fluorescent probes may be used to image cell proliferation in mice. PMID:22201533

  2. A facile synthesis of fluorescent silver nanoclusters with human ferritin as a synthetic and interfacing ligand.

    PubMed

    Lee, In Hwan; Ahn, Byungjun; Lee, Jeong Min; Lee, Chang Soo; Jung, Yongwon

    2015-05-21

    Water-soluble fluorescent silver nanoclusters (NCs) formed on biomolecule ligands have been extensively studied due to their great potential as new biocompatible fluorescent materials for biosensors. As synthetic ligands, proteins in particular can provide unique structures and functions to the assembled fluorescent silver clusters. A key challenge, however, is to develop appropriate protein ligands and synthetic approaches for cluster formation, especially using native aqueous solutions, to fully preserve the valuable properties of the protein templates. Here we report a human ferritin-templated synthesis of fluorescent silver NCs under neutral aqueous buffer conditions. The unique metal binding property of ferritin and an optimized silver ion reduction allowed us to produce highly stable fluorescent silver NCs that are steadily assembled in the cage-like ferritin proteins. The fluorescent clusters were also successfully assembled on genetically engineered ferritin with antibody-binding protein G. The resulting protein G-ferritin-silver NC complex fully retained the ferritin structure as well as the antibody binding ability. The present silver nanoclusters on ferritin (Ft-Ag NCs) also showed highly specific Cu(2+)-induced fluorescence quenching. By exploiting the large but stable nature of ferritin, we fabricated a highly robust and porous hydrogel sensor system for rapid Cu(2+) detection, where the Ft-Ag NCs were stably encapsulated in surface-bound hydrogels with large pore sizes. Our Ft-Ag NCs that are formed under native aqueous conditions will have great potential as a new fluorescent material with the high structural and functional diversities of ferritin. PMID:25848642

  3. A simple model-free method for direct assessment of fluorescent ligand binding by linear spectral summation.

    PubMed

    Gasymov, Oktay K; Abduragimov, Adil R; Glasgow, Ben J

    2014-01-01

    Fluorescent tagged ligands are commonly used to determine binding to proteins. However, bound and free ligand concentrations are not directly determined. Instead the response in a fluorescent ligand titration experiment is considered to be proportional to the extent of binding and, therefore, the maximum value of binding is scaled to the total protein concentration. Here, a simple model-free method is presented to be performed in two steps. In the first step, normalized bound and free spectra of the ligand are determined. In the second step, these spectra are used to fit composite spectra as the sum of individual components or linear spectral summation. Using linear spectral summation, free and bound 1-Anilinonaphthalene-8-Sulfonic Acid (ANS) fluorescent ligand concentrations are directly calculated to determine ANS binding to tear lipocalin (TL), an archetypical ligand binding protein. Error analysis shows that the parameters that determine bound and free ligand concentrations were recovered with high certainty. The linear spectral summation method is feasible when fluorescence intensity is accompanied by a spectral shift upon protein binding. Computer simulations of the experiments of ANS binding to TL indicate that the method is feasible when the fluorescence spectral shift between bound and free forms of the ligand is just 8 nm. Ligands tagged with environmentally sensitive fluorescent dyes, e.g., dansyl chromophore, are particularly suitable for this method. PMID:24043458

  4. A Simple Model-Free Method for Direct Assessment of Fluorescent Ligand Binding by Linear Spectral Summation

    PubMed Central

    Gasymov, Oktay K.; Abduragimov, Adil R.; Glasgow, Ben J.

    2013-01-01

    Fluorescent tagged ligands are commonly used to determine binding to proteins. However, bound and free ligand concentrations are not directly determined. Instead the response in a fluorescent ligand titration experiment is considered to be proportional to the extent of binding and, therefore, the maximum value of binding is scaled to the total protein concentration. Here, a simple model-free method is presented to be performed in two steps. In the first step, normalized bound and free spectra of the ligand are determined. In the second step, these spectra are used to fit composite spectra as the sum of individual components or linear spectral summation. Using linear spectral summation, free and bound 1-Anilinonaphthalene-8-Sulfonic Acid (ANS) fluorescent ligand concentrations are directly calculated to determine ANS binding to tear lipocalin (TL), an archetypical ligand binding protein. Error analysis shows that the parameters that determine bound and free ligand concentrations were recovered with high certainty. The linear spectral summation method is feasible when fluorescence intensity is accompanied by a spectral shift upon protein binding. Computer simulations of the experiments of ANS binding to TL indicate that the method is feasible when the fluorescence spectral shift between bound and free forms of the ligand is just 8 nm. Ligands tagged with environmentally sensitive fluorescent dyes, e.g., dansyl chromophore, are particularly suitable for this method. PMID:24043458

  5. Fluorescence-Based Strategies to Investigate the Structure and Dynamics of Aptamer-Ligand Complexes.

    PubMed

    Perez-Gonzalez, Cibran; Lafontaine, Daniel A; Penedo, J Carlos

    2016-01-01

    In addition to the helical nature of double-stranded DNA and RNA, single-stranded oligonucleotides can arrange themselves into tridimensional structures containing loops, bulges, internal hairpins and many other motifs. This ability has been used for more than two decades to generate oligonucleotide sequences, so-called aptamers, that can recognize certain metabolites with high affinity and specificity. More recently, this library of artificially-generated nucleic acid aptamers has been expanded by the discovery that naturally occurring RNA sequences control bacterial gene expression in response to cellular concentration of a given metabolite. The application of fluorescence methods has been pivotal to characterize in detail the structure and dynamics of these aptamer-ligand complexes in solution. This is mostly due to the intrinsic high sensitivity of fluorescence methods and also to significant improvements in solid-phase synthesis, post-synthetic labeling strategies and optical instrumentation that took place during the last decade. In this work, we provide an overview of the most widely employed fluorescence methods to investigate aptamer structure and function by describing the use of aptamers labeled with a single dye in fluorescence quenching and anisotropy assays. The use of 2-aminopurine as a fluorescent analog of adenine to monitor local changes in structure and fluorescence resonance energy transfer (FRET) to follow long-range conformational changes is also covered in detail. The last part of the review is dedicated to the application of fluorescence techniques based on single-molecule microscopy, a technique that has revolutionized our understanding of nucleic acid structure and dynamics. We finally describe the advantages of monitoring ligand-binding and conformational changes, one molecule at a time, to decipher the complexity of regulatory aptamers and summarize the emerging folding and ligand-binding models arising from the application of these

  6. Fluorescence-Based Strategies to Investigate the Structure and Dynamics of Aptamer-Ligand Complexes

    PubMed Central

    Perez-Gonzalez, Cibran; Lafontaine, Daniel A.; Penedo, J. Carlos

    2016-01-01

    In addition to the helical nature of double-stranded DNA and RNA, single-stranded oligonucleotides can arrange themselves into tridimensional structures containing loops, bulges, internal hairpins and many other motifs. This ability has been used for more than two decades to generate oligonucleotide sequences, so-called aptamers, that can recognize certain metabolites with high affinity and specificity. More recently, this library of artificially-generated nucleic acid aptamers has been expanded by the discovery that naturally occurring RNA sequences control bacterial gene expression in response to cellular concentration of a given metabolite. The application of fluorescence methods has been pivotal to characterize in detail the structure and dynamics of these aptamer-ligand complexes in solution. This is mostly due to the intrinsic high sensitivity of fluorescence methods and also to significant improvements in solid-phase synthesis, post-synthetic labeling strategies and optical instrumentation that took place during the last decade. In this work, we provide an overview of the most widely employed fluorescence methods to investigate aptamer structure and function by describing the use of aptamers labeled with a single dye in fluorescence quenching and anisotropy assays. The use of 2-aminopurine as a fluorescent analog of adenine to monitor local changes in structure and fluorescence resonance energy transfer (FRET) to follow long-range conformational changes is also covered in detail. The last part of the review is dedicated to the application of fluorescence techniques based on single-molecule microscopy, a technique that has revolutionized our understanding of nucleic acid structure and dynamics. We finally describe the advantages of monitoring ligand-binding and conformational changes, one molecule at a time, to decipher the complexity of regulatory aptamers and summarize the emerging folding and ligand-binding models arising from the application of these

  7. Using Fluorescence Spectroscopy to Evaluate Hill Parameters and Heterogeneity of Ligand Binding to Cytochromes P450

    NASA Astrophysics Data System (ADS)

    Marsch, Glenn A.; Carlson, Benjamin; Hansen, Jennifer; Mihelc, Elaine; Martin, Martha V.; Guengerich, F. Peter

    2009-03-01

    The cytochromes P450 (CYPs) are hemoproteins that oxidize many drugs and carcinogens. Binding interactions of two CYPs with Nile Red, pyrene, and alpha-naphthoflavone were studied using fluorescence quenching. Upon interaction with CYPs, fluorescence from pyrene excited-state dimers was quenched more efficiently than fluorescence from pyrene monomers. Quenching data was fit to the Hill equation to determine binding affinities and the Hill parameter n for the interaction of substrates with CYPs. All ligands showed strong binding to the CYPs, especially alpha-naphthoflavone, but exhibited little or no cooperativity in the interaction. Modified Stern-Volmer plots were used to confirm binding affinities, and suggested heterogeneous populations of amino acid fluorophores. Fluorescence anisotropy experiments suggest that CYP molecules tumble more rapidly when alpha-naphthoflavone is added.

  8. Resolution of ligand positions by site-directed tryptophan fluorescence in tear lipocalin.

    PubMed Central

    Gasymov, O. K.; Abduragimov, A. R.; Yusifov, T. N.; Glasgow, B. J.

    2000-01-01

    The lipocalin superfamily of proteins functions in the binding and transport of a variety of important hydrophobic molecules. Tear lipocalin is a promiscuous lipid binding member of the family and serves as a paradigm to study the molecular determinants of ligand binding. Conserved regions in the lipocalins, such as the G strand and the F-G loop, may play an important role in ligand binding and delivery. We studied structural changes in the G strand of holo- and apo-tear lipocalin using spectroscopic methods including circular dichroism analysis and site-directed tryptophan fluorescence. Apo-tear lipocalin shows the same general structural characteristics as holo-tear lipocalin including alternating periodicity of a beta-strand, orientation of amino acid residues 105, 103, 101, and 99 facing the cavity, and progressive depth in the cavity from residues 105 to 99. For amino acid residues facing the internal aspect of cavity, the presence of a ligand is associated with blue shifted spectra. The collisional rate constants indicate that these residues are not less exposed to solvent in holo-tear lipocalin than in apo-tear lipocalin. Rather the spectral blue shifts may be accounted for by a ligand induced rigidity in holo-TL. Amino acid residues 94 and 95 are consistent with positions in the F-G loop and show greater exposure to solvent in the holo- than the apo-proteins. These findings are consistent with the general hypothesis that the F-G loop in the holo-proteins of the lipocalin family is available for receptor interactions and delivery of ligands to specific targets. Site-directed tryptophan fluorescence was used in combination with a nitroxide spin labeled fatty acid analog to elucidate dynamic ligand interactions with specific amino acid residues. Collisional quenching constants of the nitroxide spin label provide evidence that at least three amino acids of the G strand residues interact with the ligand. Stern-Volmer plots are inconsistent with a ligand that is

  9. Resolution of ligand positions by site-directed tryptophan fluorescence in tear lipocalin.

    PubMed

    Gasymov, O K; Abduragimov, A R; Yusifov, T N; Glasgow, B J

    2000-02-01

    The lipocalin superfamily of proteins functions in the binding and transport of a variety of important hydrophobic molecules. Tear lipocalin is a promiscuous lipid binding member of the family and serves as a paradigm to study the molecular determinants of ligand binding. Conserved regions in the lipocalins, such as the G strand and the F-G loop, may play an important role in ligand binding and delivery. We studied structural changes in the G strand of holo- and apo-tear lipocalin using spectroscopic methods including circular dichroism analysis and site-directed tryptophan fluorescence. Apo-tear lipocalin shows the same general structural characteristics as holo-tear lipocalin including alternating periodicity of a beta-strand, orientation of amino acid residues 105, 103, 101, and 99 facing the cavity, and progressive depth in the cavity from residues 105 to 99. For amino acid residues facing the internal aspect of cavity, the presence of a ligand is associated with blue shifted spectra. The collisional rate constants indicate that these residues are not less exposed to solvent in holo-tear lipocalin than in apo-tear lipocalin. Rather the spectral blue shifts may be accounted for by a ligand induced rigidity in holo-TL. Amino acid residues 94 and 95 are consistent with positions in the F-G loop and show greater exposure to solvent in the holo- than the apo-proteins. These findings are consistent with the general hypothesis that the F-G loop in the holo-proteins of the lipocalin family is available for receptor interactions and delivery of ligands to specific targets. Site-directed tryptophan fluorescence was used in combination with a nitroxide spin labeled fatty acid analog to elucidate dynamic ligand interactions with specific amino acid residues. Collisional quenching constants of the nitroxide spin label provide evidence that at least three amino acids of the G strand residues interact with the ligand. Stern-Volmer plots are inconsistent with a ligand that is

  10. Ligand replacement-induced fluorescence switch of quantum dots for ultrasensitive detection of organophosphorothioate pesticides.

    PubMed

    Zhang, Kui; Mei, Qingsong; Guan, Guijian; Liu, Bianhua; Wang, Suhua; Zhang, Zhongping

    2010-11-15

    The development of a simple and on-site assay for the detection of organophosphorus pesticed residues is very important for food safety and exosystem protection. This paper reports the surface coordination-originated fluorescence resonance energy transfer (FRET) of CdTe quantum dots (QDs) and a simple ligand-replacement turn-on mechanism for the highly sensitive and selective detection of organophosphorothioate pesticides. It has been demonstrated that coordination of dithizone at the surface of CdTe QDs in basic media can strongly quench the green emission of CdTe QDs by a FRET mechanism. Upon the addition of organophosphorothioate pesticides, the dithizone ligands at the CdTe QD surface are replaced by the hydrolyzate of the organophosphorothioate, and hence the fluorescence is turned on. The fluorescence turn on is immediate, and the limit of detection for chlorpyrifos is as low as ∼0.1 nM. Two consecutive linear ranges allow a wide determination of chlorpyrifos concentrations from 0.1 nM to 10 μM. Importantly, the fluorescence turn-on chemosensor can directly detect chlorpyrifos residues in apples at a limit of 5.5 ppb, which is under the maximum residue limit allowed by the U.S. Environmental Protection Agency. The very simple strategy reported here should facilitate the development of fluorescence turn-on chemosensors for chemo/biodetection. PMID:20973515

  11. Ligand-induced evolution of intrinsic fluorescence and catalytic activity from cobalt ferrite nanoparticles.

    PubMed

    Pal, Monalisa; Kundu, Anirban; Rakshit, Rupali; Mandal, Kalyan

    2015-06-01

    To develop CoFe(2)O(4) as magneto-fluorescent nanoparticles (NPs) for biomedical applications, it would be advantageous to identify any intrinsic fluorescence of this important magnetic material by simply adjusting the surface chemistry of the NPs themselves. Herein, we demonstrate that intrinsic multicolor fluorescence, covering the whole visible region, can be induced by facile functionalization of CoFe(2)O(4) NPs with Na-tartrate. Moreover, the functionalized CoFe(2)O(4) NPs also show unprecedented catalytic efficiency in the degradation of both biologically and environmentally harmful dyes, pioneering the potential application of these NPs in therapeutics and wastewater treatment. Detailed investigation through various spectroscopic tools unveils the story behind the emergence of this unique optical property of CoFe(2)O(4) NPs upon functionalization with tartrate ligands. We believe our developed multifunctional CoFe(2)O(4) NPs hold great promise for advanced biomedical and technological applications. PMID:25867626

  12. Aqueous Growth of Gold Clusters with Tunable Fluorescence Using Photochemically Modified Lipoic Acid-Based Ligands.

    PubMed

    Mishra, Dinesh; Aldeek, Fadi; Lochner, Eric; Palui, Goutam; Zeng, Birong; Mackowski, Sebastian; Mattoussi, Hedi

    2016-06-28

    We report a one-phase aqueous growth of fluorescent gold nanoclusters (AuNCs) with tunable emission in the visible spectrum, using a ligand scaffold that is made of poly(ethylene glycol) segment appended with a metal coordinating lipoic acid at one end and a functional group at the other end. This synthetic scheme exploits the ability of the UV-induced photochemical transformation of LA-based ligands to provide DHLA and other thiol byproducts that exhibit great affinity to metal nanoparticles, obviating the need for chemical reduction of the dithiolane ring using classical reducing agents. The influence of various experimental conditions, including the photoirradiation time, gold precursor-to-ligand molar ratios, time of reaction, temperature, and the medium pH, on the growth of AuNCs has been systematically investigated. The photophysical properties, size, and structural characterization were carried out using UV-vis absorption and fluorescence spectroscopy, TEM, DOSY-NMR, and X-ray photoelectron spectroscopy. The hydrodynamic size (RH) obtained by DOSY-NMR indicates that the size of these clusters follows the trend anticipated from the absorption and PL data, with RH(red) > RH(yellow) > RH(blue). The tunable emission and size of these gold nanoclusters combined with their high biocompatibility would make them greatly promising for potential use in imaging and sensing applications. PMID:27254320

  13. Design of a Water Soluble Fluorescent 3-Hydroxy-4-Pyridinone Ligand Active at Physiological pH Values.

    PubMed

    Leite, Andreia; Silva, Ana M G; Coutinho, Catarina; Cunha-Silva, Luís; de Castro, Baltazar; Rangel, Maria

    2016-09-01

    In the present work we report the structure and the spectroscopic characterization of a new fluorescent 3-hydroxy-4-pyridinone ligand D-3,4-HPO. The synthesis of the compound was performed in two steps, which involve the reaction of the commercially available fluorophore dansyl chloride with a 3-hydroxy-4-pyridinone chelating unit and further deprotection. The new fluorescent chelator was characterized in the solid state by single-crystal X-ray diffraction and in solution by NMR, MS, absorption and fluorescence spectroscopies. The analysis of the variation of the absorption spectrum with pH allowed the determination of four pK a values (pK a1  = 3.50, pK a2  = 4.50, pK a3  = 9.60, pK a4  = 10.20) and establishment of the corresponding distribution diagram. The study of the fluorescence properties of the ligand show that in the pH range between 4 and 9 the fluorescence intensity is constant and has its maximum value thus allowing its further use at physiological pH values. The interaction of the ligand with copper(II) was accessed by fluorescence spectroscopy in MOPS buffer and the results show that the presence of copper(II) quenches the fluorescence of the ligand in ca 94 % at a ligand: metal ratio of 2:1. The latter result is consistent with the formation of a copper(II) complex with the bidentate ligand, as confirmed by the EPR spectroscopy. Graphical Abstract New water soluble fluorescent ligand active at physiological pH values. PMID:27357392

  14. A biosensor for theophylline based on fluorescence detection of ligand-induced hammerhead ribozyme cleavage.

    PubMed Central

    Sekella, Phillip T; Rueda, David; Walter, Nils G

    2002-01-01

    Recently, Breaker and coworkers engineered hammerhead ribozymes that rearrange from a catalytically inactive to an active conformation upon allosteric binding of a specific ligand. To monitor cleavage activity in real time, we have coupled a donor-acceptor fluorophore pair to the termini of the substrate RNA of such a hammerhead ribozyme, modified to cleave in trans in the presence of the bronchodilator theophylline. In the intact substrate, the fluorophores interact by fluorescence resonance energy transfer (FRET). The specific FRET signal breaks down as the effector ligand binds, the substrate is cleaved, and the products dissociate, with a rate constant dependent on the concentration of the ligand. Our biosensor cleaves substrate at 0.46 min(-1) in 1 mM theophylline and 0.04 min(-1) without effector, and discriminates against caffeine, a structural relative of theophylline. We have measured the theophylline-dependence profile of this biosensor, showing that concentrations as low as 1 microM can be distinguished from background. To probe the mechanism of allosteric regulation, a single nucleotide in the communication domain between the catalytic and ligand-binding domains was mutated to destabilize the inactive conformation of the ribozyme. As predicted, this mutant shows the same activity (0.3 min(-1)) in the presence and absence of theophylline. Additionally, time-resolved FRET measurements on the biosensor ribozyme in complex with a noncleavable substrate analog reveal no significant changes in fluorophore distance distribution upon binding of effector. PMID:12403463

  15. A Fluorescence Displacement Assay for Antidepressant Drug Discovery Based on Ligand-Conjugated Quantum Dots

    SciTech Connect

    Chang, Jerry; Tomlinson, Ian; Warnement, Michael; Iwamoto, Hideki

    2011-01-01

    The serotonin (5-hydroxytryptamine, 5-HT) transporter (SERT) protein plays a central role in terminating 5-HT neurotransmission and is the most important therapeutic target for the treatment of major depression and anxiety disorders. We report an innovative, versatile, and target-selective quantum dot (QD) labeling approach for SERT in single Xenopus oocytes that can be adopted as a drug-screening platform. Our labeling approach employs a custom-made, QD-tagged indoleamine derivative ligand, IDT318, that is structurally similar to 5-HT and accesses the primary binding site with enhanced human SERT selectivity. Incubating QD-labeled oocytes with paroxetine (Paxil), a high-affinity SERT-specific inhibitor, showed a concentration- and time-dependent decrease in QD fluorescence, demonstrating the utility of our approach for the identification of SERT modulators. Furthermore, with the development of ligands aimed at other pharmacologically relevant targets, our approach may potentially form the basis for a multitarget drug discovery platform.

  16. Development of sigma-1 (σ1) receptor fluorescent ligands as versatile tools to study σ1 receptors✩

    PubMed Central

    Abate, Carmen; Riganti, Chiara; Pati, Maria Laura; Ghigo, Dario; Berardi, Francesco; Mavlyutov, Timur; Guo, Lian-Wang; Ruoho, Arnold

    2016-01-01

    Despite their controversial physiology, sigma-1 (σ1) receptors are intriguing targets for the development of therapeutic agents for central nervous system diseases. With the aim of providing versatile pharmacological tools to study σ1 receptors, we developed three σ1 fluorescent tracers by functionalizing three well characterized σ1 ligands with a fluorescent tag. A good compromise between σ1 binding affinity and fluorescent properties was reached, and the σ1 specific targeting of the novel tracers was demonstrated by confocal microscopy and flow cytometry. These novel ligands were also successfully used in competition binding studies by flow cytometry, showing their utility in nonradioactive binding assays as an alternative strategy to the more classical radioligand binding assays. To the best of our knowledge these are the first σ1 fluorescent ligands to be developed and successfully employed in living cells, representing promising tools to strengthen σ1 receptors related studies. PMID:26717207

  17. Bodilisant—A Novel Fluorescent, Highly Affine Histamine H3 Receptor Ligand

    PubMed Central

    2012-01-01

    A piperidine-based lead structure for the human histamine H3 receptor (hH3R) was coupled with the BODIPY fluorophore and resulted in a strong green fluorescent (quantum yield, 0.92) hH3R ligand with affinity in the nanomolar concentration range (Ki hH3R = 6.51 ± 3.31 nM), named Bodilisant. Screening for affinities at histamine and dopamine receptor subtypes showed high hH3R preference. Bodilisant was used for visualization of hH3R in hH3R overexpressing HEK-293 cells with fluorescence confocal laser scanning microscopy. In addition, in native human brain tissues, Bodilisant showed clear and displaceable images of labeled hH3R. PMID:24900647

  18. A fluorescent reporter detects details of aromatic ligand interference in drug-binding sites of human serum albumin.

    PubMed

    Dobretsov, Gennady; Smolina, Natalia; Syrejshchikova, Tatiana; Brilliantova, Varvara; Uzbekov, Marat

    2016-09-01

    Human serum albumin (HSA) transports many ligands including small aromatic molecules: metabolites, drugs etc. Phenylbutazone is an anti-inflammatory drug, which binds to the drug-binding site I of HSA. Its interaction with this site has been studied using a fluorescent dye, CAPIDAN, whose fluorescence in serum originates from HSA and is sensitive to the changes in HSA site I in some diseases. Its fluorescence in HSA solutions is strongly suppressed by phenylbutazone. This phenomenon seems to be a basic sign of a simple drug-dye competition. However, a more detailed study of the time-resolved fluorescence decay of CAPIDAN has shown that phenylbutazone lowers fluorescence without changing the total amount of bound dye. In brief, the HSA-bound dye forms three populations due to three types of environment at the binding sites. The first two populations probably have a rather strong Coulomb interaction with the positive charge of residues Arginine 218 or Arginine 222 in site I and are responsible for approximately 90% of the total fluorescence. Phenylbutazone blocks this interaction and therefore lowers this fluorescence. At the same time the binding of the third population increases considerably in the presence of phenylbutazone, and, as a result, the actual number of bound dye molecules remains almost unchanged despite the ligand competition. So, time resolved fluorescence of the reporter allows to observe details of interactions and interference of aromatic ligands in drug binding site I of HSA both in isolated HSA and in serum. PMID:27318089

  19. First fluorescence spectroscopic investigation of Am(III) complexation with an organic carboxylic ligand, pyromellitic acid.

    PubMed

    Barkleit, Astrid; Geipel, Gerhard; Acker, Margret; Taut, Steffen; Bernhard, Gert

    2011-01-01

    For the first time Am(III) complexation with a small organic ligand could be identified and characterized with time-resolved laser-induced fluorescence spectroscopy (TRLFS) at room temperature and trace metal concentration. With pyromellitic acid (1,2,4,5-benzene-tetracarboxylic acid, BTC) as ligand spectroscopic characteristics for the Am-BTC complex system were determined at pH 5.0, an ionic strength of 0.1 M (NaClO4) and room temperature. The fluorescence lifetimes were determined to be 23.2±2.2 ns for Am3+(aq) and 27.2±1.2 ns for the Am-BTC 1:1 complex; the emission maximum for the 5D1-(7)F1 transition is 691 nm for both species. The complex stability constant for the Am-BTC 1:1 complex was calculated to be logβ110=5.42±0.16. PMID:20943431

  20. Highly Fluorescent Group 13 Metal Complexes with Cyclic, Aromatic Hydroxamic Acid Ligands

    SciTech Connect

    Seitz, Michael; Moore, Evan G.; Raymond, Kenneth N.

    2008-02-11

    The neutral complexes of two ligands based on the 1-oxo-2-hydroxy-isoquinoline (1,2-HOIQO) motif with group 13 metals (Al, Ga, In) show bright blue-violet luminescence in organic solvents. The corresponding transition can be attributed to ligand-centered singlet emission, characterized by a small Stokes shifts of only a few nm combined with lifetimes in the range between 1-3 ns. The fluorescence efficiency is high, with quantum yields of up to 37% in benzene solution. The crystal structure of one of the indium(III) complexes (trigonal space group R-3, a = b = 13.0384(15) {angstrom}, c = 32.870(8) {angstrom}, ? = {beta} = 90{sup o}, {gamma} = 120{sup o}, V = 4839.3(14) {angstrom}{sup 3}, Z = 6) shows a six-coordinate geometry around the indium center which is close to trigonal-prismatic, with a twist angle between the two trigonal faces of 20.7{sup o}. Time-dependent density functional theory (TD-DFT) calculations (Al and Ga: B3LYP/6-31G(d)); In: B3LYP/LANL2DZ of the fac and mer isomers with one of the two ligands indicate that there is no clear preference for either one of the isomeric forms of the metal complexes. In addition, the metal centers do not have a significant influence on the electronic structure, and as a consequence, on the predominant intraligand optical transitions.

  1. Structural and biophysical characterisation of G protein-coupled receptor ligand binding using resonance energy transfer and fluorescent labelling techniques.

    PubMed

    Ward, Richard J; Milligan, Graeme

    2014-01-01

    The interaction between ligands and the G protein-coupled receptors (GPCRs) to which they bind has long been the focus of intensive investigation. The signalling cascades triggered by receptor activation, due in most cases to ligand binding, are of great physiological and medical importance; indeed, GPCRs are targeted by in excess of 30% of small molecule therapeutic medicines. Attempts to identify further pharmacologically useful GPCR ligands, for receptors with known and unknown endogenous ligands, continue apace. In earlier days direct assessment of such interactions was restricted largely to the use of ligands incorporating radioactive isotope labels as this allowed detection of the ligand and monitoring its interaction with the GPCR. This use of such markers has continued with the development of ligands labelled with fluorophores and their application to the study of receptor-ligand interactions using both light microscopy and resonance energy transfer techniques, including homogenous time-resolved fluorescence resonance energy transfer. Details of ligand-receptor interactions via X-ray crystallography are advancing rapidly as methods suitable for routine production of substantial amounts and stabilised forms of GPCRs have been developed and there is hope that this may become as routine as the co-crystallisation of serine/threonine kinases with ligands, an approach that has facilitated widespread use of rapid structure-based ligand design. Conformational changes involved in the activation of GPCRs, widely predicted by biochemical and biophysical means, have inspired the development of intramolecular FRET-based sensor forms of GPCRs designed to investigate the events following ligand binding and resulting in a signal propagation across the cell membrane. Finally, a number of techniques are emerging in which ligand-GPCR binding can be studied in ways that, whilst indirect, are able to monitor its results in an unbiased and integrated manner. This article is part

  2. On the origin of non-exponential fluorescence decays in enzyme-ligand complex

    NASA Astrophysics Data System (ADS)

    Wlodarczyk, Jakub; Kierdaszuk, Borys

    2004-05-01

    Complex fluorescence decays have usually been analyzed with the aid of a multi-exponential model, but interpretation of the individual exponential terms has not been adequately characterized. In such cases the intensity decays were also analyzed in terms of the continuous lifetime distribution as a consequence of an interaction of fluorophore with environment, conformational heterogeneity or their dynamical nature. We show that non-exponential fluorescence decay of the enzyme-ligand complexes may results from time dependent energy transport. The latter, to our opinion, may be accounted for by electron transport from the protein tyrosines to their neighbor residues. We introduce the time-dependent hopping rate in the form v(t)~(a+bt)-1. This in turn leads to the luminescence decay function in the form I(t)=Ioexp(-t/τ1)(1+lt/γτ2)-γ. Such a decay function provides good fits to highly complex fluorescence decays. The power-like tail implies the time hierarchy in migration energy process due to the hierarchical energy-level structure. Moreover, such a power-like term is a manifestation of so called Tsallis nonextensive statistic and is suitable for description of the systems with long-range interactions, memory effect as well as with fluctuations of characteristic lifetime of fluorescence. The proposed decay function was applied in analysis of fluorescence decays of tyrosine protein, i.e. the enzyme purine nucleoside phosphorylase from E. coli in a complex with formycin A (an inhibitor) and orthophosphate (a co-substrate).

  3. Red fluorescent turn-on ligands for imaging and quantifying G protein-coupled receptors in living cells.

    PubMed

    Karpenko, Iuliia A; Kreder, Rémy; Valencia, Christel; Villa, Pascal; Mendre, Christiane; Mouillac, Bernard; Mély, Yves; Hibert, Marcel; Bonnet, Dominique; Klymchenko, Andrey S

    2014-02-10

    Classical fluorescence-based approaches to monitor ligand-protein interactions are generally hampered by the background signal of unbound ligand, which must be removed by tedious washing steps. To overcome this major limitation, we report here the first red fluorescent turn-on probes for a G protein-coupled receptor (oxytocin receptor) at the surface of living cells. The peptide ligand carbetocin was conjugated to one of the best solvatochromic (fluorogenic) dyes, Nile Red, which turns on emission when reaching the hydrophobic environment of the receptor. We showed that the incorporation of hydrophilic octa(ethylene glycol) linker between the pharmacophore and the dye minimized nonspecific interaction of the probe with serum proteins and lipid membranes, thus ensuring receptor-specific turn-on response. The new ligand was successfully applied for background-free imaging and quantification of oxytocin receptors in living cells. PMID:24449564

  4. Effect of Fluorescently Labeling Protein Probes on Kinetics of Protein-Ligand Reactions

    PubMed Central

    Sun, Y.S.; Landry, J.P.; Fei, Y.Y.; Luo, J.T.; Wang, X.B.; Lam, K.S.

    2009-01-01

    We studied the effect of fluorescently labeling proteins on protein-ligand reactions. Un-labeled ligands (streptavidin-binding peptides and rabbit immunoglobulin G (IgG) as antigen targets) are immobilized on epoxy-functionalized glass slides. Unlabeled and Cy3-labeled protein probes from the same batch (streptavidin and goat antibodies) subsequently react with the surface-immobilized targets. By monitoring in situ the surface mass density change using an oblique-incidence reflectivity difference scanning microscope (a label-free detector), we measured kon and koff for streptavidin-peptide reactions and antibody-antigen reaction. We found that (1) equilibrium dissociation constants, defined as KD = koff/kon, for streptavidin-peptide reactions increases by a factor of 3 ~ 4 when the solution-phase streptavidin is labeled with Cy3 dye; and (2) KD for reactions of solution-phase goat anti-rabbit antibodies with rabbit IgG targets also change significantly when the goat antibodies are labeled with Cy3 dye. PMID:18991423

  5. Effect of fluorescently labeling protein probes on kinetics of protein-ligand reactions.

    PubMed

    Sun, Y S; Landry, J P; Fei, Y Y; Zhu, X D; Luo, J T; Wang, X B; Lam, K S

    2008-12-01

    We studied the effect of fluorescently labeling proteins on protein-ligand reactions. Unlabeled ligands (streptavidin-binding peptides and rabbit immunoglobulin G (IgG) as antigen targets) are immobilized on epoxy-functionalized glass slides. Unlabeled and Cy3-labeled protein probes from the same batch (streptavidin and goat antibodies) subsequently react with the surface-immobilized targets. By monitoring in situ the surface mass density change using an oblique-incidence reflectivity difference scanning microscope (a label-free detector), we measured k(on) and k(off) for streptavidin-peptide reactions and antibody-antigen reaction. We found that (1) equilibrium dissociation constants, defined as K(D) = k(off)/k(on), for streptavidin-peptide reactions increases by a factor of 3-4 when the solution-phase streptavidin is labeled with Cy3 dye and (2) K(D) for reactions of solution-phase goat anti-rabbit antibodies with rabbit IgG targets also change significantly when the goat antibodies are labeled with Cy3 dye. PMID:18991423

  6. A helicase assay based on the displacement of fluorescent, nucleic acid-binding ligands.

    PubMed Central

    Eggleston, A K; Rahim, N A; Kowalczykowski, S C

    1996-01-01

    We have developed a new helicase assay that overcomes many limitations of other assays used to measure this activity. This continuous, kinetic assay is based on the displacement of fluorescent dyes from dsDNA upon DNA unwinding. These ligands exhibit significant fluorescence enhancement when bound to duplex nucleic acids and serve as the reporter molecules of DNA unwinding. We evaluated the potential of several dyes [acridine orange, ethidium bromide, ethidium homodimer, bis-benzimide (DAPI), Hoechst 33258 and thiazole orange] to function as suitable reporter molecules and demonstrate that the latter three dyes can be used to monitor the helicase activity of Escherichia coli RecBCD enzyme. Both the binding stoichiometry of RecBCD enzyme for the ends of duplex DNA and the apparent rate of unwinding are not significantly perturbed by two of these dyes. The effects of temperature and salt concentration on the rate of unwinding were also examined. We propose that this dye displacement assay can be readily adapted for use with other DNA helicases, with RNA helicases, and with other enzymes that act on nucleic acids. PMID:8614617

  7. A Microbead Supported Membrane-Based Fluorescence Imaging Assay Reveals Intermembrane Receptor-Ligand Complex Dimension with Nanometer Precision.

    PubMed

    Biswas, Kabir H; Groves, Jay T

    2016-07-01

    Receptor-ligand complexes spanning a cell-cell interface inevitably establish a preferred intermembrane spacing based on the molecular dimensions and orientation of the complexes. This couples molecular binding events to membrane mechanics and large-scale spatial organization of receptors on the cell surface. Here, we describe a straightforward, epi-fluorescence-based method to precisely determine intermembrane receptor-ligand dimension at adhesions established by receptor-ligand binding between apposed membranes in vitro. Adhesions were reconstituted between planar and silica microbead supported membranes via specific interaction between cognate receptor/ligand pairs (EphA2/EphrinA1 and E-cadherin/anti-E-cadherin antibody). Epi-fluorescence imaging of the ligand enrichment zone in the supported membrane beneath the adhering microbead, combined with a simple geometrical interpretation, proves sufficient to estimate intermembrane receptor-ligand dimension with better than 1 nm precision. An advantage of this assay is that no specialized equipment or imaging methods are required. PMID:27264296

  8. Uranium(VI) coordination polymers with pyromellitate ligand: Unique 1D channel structures and diverse fluorescence

    SciTech Connect

    Zhang, Yingjie; Bhadbhade, Mohan; Karatchevtseva, Inna; Price, Jason R.; Liu, Hao; Zhang, Zhaoming; Kong, Linggen; Čejka, Jiří; Lu, Kim; Lumpkin, Gregory R.

    2015-03-15

    Three new coordination polymers of uranium(VI) with pyromellitic acid (H{sub 4}btca) have been synthesized and structurally characterized. (ED)[(UO{sub 2})(btca)]·(DMSO)·3H{sub 2}O (1) (ED=ethylenediammonium; DMSO=dimethylsulfoxide) has a lamellar structure with intercalation of ED and DMSO. (NH{sub 4}){sub 2}[(UO{sub 2}){sub 6}O{sub 2}(OH){sub 6}(btca)]·~6H{sub 2}O (2) has a 3D framework built from 7-fold coordinated uranyl trinuclear units and btca ligands with 1D diamond-shaped channels (~8.5 Å×~8.6 Å). [(UO{sub 2}){sub 2}(H{sub 2}O)(btca)]·4H{sub 2}O (3) has a 3D network constructed by two types of 7-fold coordinated uranium polyhedron. The unique μ{sub 5}-coordination mode of btca in 3 enables the formation of 1D olive-shaped large channels (~4.5 Å×~19 Å). Vibrational modes, thermal stabilities and fluorescence properties have been investigated. - Graphical abstract: Table of content: three new uranium(VI) coordination polymers with pyromellitic acid (H{sub 4}btca) have been synthesized via room temperature and hydrothermal synthesis methods, and structurally characterized. Two to three dimensional (3D) frameworks are revealed. All 3D frameworks have unique 1D large channels. Their vibrational modes, thermal stabilities and photoluminescence properties have been investigated. - Highlights: • Three new coordination polymers of U(VI) with pyromellitic acid (H{sub 4}btca). • Structures from a 2D layer to 3D frameworks with unique 1D channels. • Unusual µ{sub 5}-(η{sub 1}:η{sub 2}:η{sub 1}:η{sub 2:}η{sub 1}) coordination mode of btca ligand. • Vibrational modes, thermal stabilities and luminescent properties reported.

  9. Highly sensitive and selective fluorescence detection of copper (II) ion based on multi-ligand metal chelation.

    PubMed

    Zhang, Shan; Yu, Tao; Sun, Mingtai; Yu, Huan; Zhang, Zhongping; Wang, Suhua; Jiang, Hui

    2014-08-01

    A fluorescent probe was synthesized and demonstrated to be highly selective and sensitive in the reaction with copper (II) ion, generating a large variation of the fluorescence intensity in a dose-response manner. The probe contains a dansyl moiety as fluorophore and a multidentate ligand for copper (II) ion recognition. The reaction of the molecular probe with copper (II) ion proceeds rapidly and irreversibly in a 1 to 1 stoichiometric way, leading to the production of stable copper (II) complex, which subsequently results in the quenching of fluorescence. The detection limit for copper (II) ion was measured to be about 2ppb. It was also shown that the probe has high selectivity for copper (II) ion and good anti-interference ability against other transition metal ions. The herein reported very simple and reliable fluorescence probe could be employed for copper (II) ion detection in many aspects. PMID:24881551

  10. Real-time Monitoring of Ligand-receptor Interactions with Fluorescence Resonance Energy Transfer

    PubMed Central

    Dogra, Navneet; Reyes, Julia C.; Garg, Nishi; Kohli, Punit

    2012-01-01

    FRET is a process whereby energy is non-radiatively transferred from an excited donor molecule to a ground-state acceptor molecule through long-range dipole-dipole interactions1. In the present sensing assay, we utilize an interesting property of PDA: blue-shift in the UV-Vis electronic absorption spectrum of PDA (Figure 1) after an analyte interacts with receptors attached to PDA2,3,4,7. This shift in the PDA absorption spectrum provides changes in the spectral overlap (J) between PDA (acceptor) and rhodamine (donor) that leads to changes in the FRET efficiency. Thus, the interactions between analyte (ligand) and receptors are detected through FRET between donor fluorophores and PDA. In particular, we show the sensing of a model protein molecule streptavidin. We also demonstrate the covalent-binding of bovine serum albumin (BSA) to the liposome surface with FRET mechanism. These interactions between the bilayer liposomes and protein molecules can be sensed in real-time. The proposed method is a general method for sensing small chemical and large biochemical molecules. Since fluorescence is intrinsically more sensitive than colorimetry, the detection limit of the assay can be in sub-nanomolar range or lower8. Further, PDA can act as a universal acceptor in FRET, which means that multiple sensors can be developed with PDA (acceptor) functionalized with donors and different receptors attached on the surface of PDA liposomes. PMID:22929922

  11. Beyond radio-displacement techniques for Identification of CB1 Ligands: The First Application of a Fluorescence-quenching Assay

    PubMed Central

    Bruno, Agostino; Lembo, Francesca; Novellino, Ettore; Stornaiuolo, Mariano; Marinelli, Luciana

    2014-01-01

    Cannabinoid type 1 Receptor (CB1) belongs to the GPCR family and it has been targeted, so far, for the discovery of drugs aimed at the treatment of neuropathic pain, nausea, vomit, and food intake disorders. Here, we present the development of the first fluorescent assay enabling the measurement of kinetic binding constants for CB1orthosteric ligands. The assay is based on the use of T1117, a fluorescent analogue of AM251. We prove that T1117 binds endogenous and recombinant CB1 receptors with nanomolar affinity. Moreover, T1117 binding to CB1 is sensitive to the allosteric ligand ORG27569 and thus it is applicable to the discovery of new allosteric drugs. The herein presented assay constitutes a sustainable valid alternative to the expensive and environmental impacting radiodisplacement techniques and paves the way for an easy, fast and cheap high-throughput drug screening toward CB1 for identification of new orthosteric and allosteric modulators. PMID:24441508

  12. Investigations into the bovine serum albumin binding and fluorescence properties of Tb (III) complex of a novel 8-hydroxyquinoline ligand

    NASA Astrophysics Data System (ADS)

    Zhao, Mingming; Tang, Ruiren; Xu, Shuai

    2015-01-01

    A novel ligand, 2-methyl-6-(8-quinolinyl)-dicarboxylate pyridine (L), and its corresponding Tb (III) complex, Na4Tb(L)2Cl4·3H2O, were successfully prepared and characterized. The luminescence spectra showed that the ligand L was an efficient sensitizer for Tb (III) luminescence. The interaction of the complex with bovine serum albumin (BSA) was investigated through fluorescence spectroscopy under physiological conditions. The Stern-Volmer analysis indicated that the fluorescence quenching was resulted from static mechanism. The binding sites (n) approximated 1.0 and this meant that interaction of Na4Tb(L)2Cl4·3H2O with BSA had single binding site. The results showed van der Waals interactions and hydrogen bonds played major roles in the binding reaction. Furthermore, circular dichroism (CD) spectra indicated that the conformation of BSA was changed.

  13. Beyond radio-displacement techniques for identification of CB1 ligands: the first application of a fluorescence-quenching assay.

    PubMed

    Bruno, Agostino; Lembo, Francesca; Novellino, Ettore; Stornaiuolo, Mariano; Marinelli, Luciana

    2014-01-01

    Cannabinoid type 1 Receptor (CB1) belongs to the GPCR family and it has been targeted, so far, for the discovery of drugs aimed at the treatment of neuropathic pain, nausea, vomit, and food intake disorders. Here, we present the development of the first fluorescent assay enabling the measurement of kinetic binding constants for CB1 orthosteric ligands. The assay is based on the use of T1117, a fluorescent analogue of AM251. We prove that T1117 binds endogenous and recombinant CB1 receptors with nanomolar affinity. Moreover, T1117 binding to CB1 is sensitive to the allosteric ligand ORG27569 and thus it is applicable to the discovery of new allosteric drugs. The herein presented assay constitutes a sustainable valid alternative to the expensive and environmental impacting radiodisplacement techniques and paves the way for an easy, fast and cheap high-throughput drug screening toward CB1 for identification of new orthosteric and allosteric modulators. PMID:24441508

  14. Fluorescence spectroscopic analysis of ligand binding to kringle 1 + 2 + 3 and kringle 1 fragments from human plasminogen.

    PubMed

    Matsuka, Y V; Novokhatny, V V; Kudinov, S A

    1990-05-31

    The ligand binding of kringle 1 + 2 + 3 and kringle 1 from human plasminogen has been investigated by fluorescence spectroscopy. Analysis of fluorescence titration of kringle 1 + 2 + 3 with 6-aminohexanoic acid shows that this fragment, besides the high-affinity lysine-binding site with Kd = 2.9 microM, contains two additional lysine-binding sites which differ in binding strength (Kd = 28 microM and Kd = 220 microM). This strongly suggests the existence of a lysine-binding site in each of the first three kringles. 6-Aminohexanoic acid, pentylamine, pentanoic acid and arginine were used for investigation of the ligand specificity of isolated kringle 1 prepared by pepsin hydrolysis of kringle 1 + 2 + 3. It has been established that kringle 1 has high affinity to 6-aminohexanoicacid, pentylamine and arginine (Kd values are 3.2 microM, 4.8 microM and 4.3 microM, respectively). At the same time pentanoic acid did not bind with kringle 1. These facts indicate, firstly, a broad ligand specificity of kringle 1 and, secondly, the paramount importance of the positively charged group of the ligand for its interaction with lysine-binding site of this kringle. PMID:2163837

  15. Ligand-induced folding of the thiM TPP riboswitch investigated by a structure-based fluorescence spectroscopic approach

    PubMed Central

    Lang, Kathrin; Rieder, Renate; Micura, Ronald

    2007-01-01

    Riboswitches are genetic control elements within non-coding regions of mRNA. They consist of a metabolite-sensitive aptamer and an adjoining expression platform. Here, we describe ligand-induced folding of a thiamine pyrophosphate (TPP) responsive riboswitch from Escherichia coli thiM mRNA, using chemically labeled variants. Referring to a recent structure determination of the TPP/aptamer complex, each variant was synthesized with a single 2-aminopurine (AP) nucleobase replacement that was selected to monitor formation of tertiary interactions of a particular region during ligand binding in real time by fluorescence experiments. We have determined the rate constants for conformational adjustment of the individual AP sensors. From the 7-fold differentiation of these constants, it can be deduced that tertiary contacts between the two parallel helical domains (P2/J3-2/P3/L3 and P4/P5/L5) that grip the ligand's ends in two separate pockets, form significantly faster than the function-critical three-way junction with stem P1 fully developed. Based on these data, we characterize the process of ligand binding by an induced fit of the RNA and propose a folding model of the TPP riboswitch aptamer. For the full-length riboswitch domain and for shorter constructs that represent transcriptional intermediates, we have additionally evaluated ligand-induced folding via AP-modified variants and provide insights into the sequential folding pathway that involves a finely balanced equilibrium of secondary structures. PMID:17693433

  16. In Vivo Stable Tumor-Specific Painting in Various Colors Using Dehalogenase-Based Protein-Tag Fluorescent Ligands

    PubMed Central

    Kosaka, Nobuyuki; Ogawa, Mikako; Choyke, Peter L.; Karassina, Natasha; Corona, Cesear; McDougall, Mark; Lynch, David; Hoyt, Clifford; Levenson, Richard; Los, Georgyi V.; Kobayashi, Hisataka

    2010-01-01

    In vivo fluorescence cancer imaging is an important tool in understanding tumor growth and therapeutic monitoring and can be performed either with endogenously produced fluorescent proteins or exogenously introduced fluorescent probes bound to targeting molecules. However, endogenous fluorescence proteins cannot be altered after transfection, thus requiring rederivation of cell lines for each desired color, while exogenously targeted fluorescence probes are limited by the heterogeneous expression of naturally occurring cellular targets. In this study, we adapted the dehalogenase-based protein-Tag (HaloTag) system to in vivo cancer imaging. By introducing highly expressed HaloTag receptors (HaloTagR) in cancer cells coupled with an externally injected a range of fluorophore-conjugated dehalogenase-reactive sequences. Tumor nodules arising from a single transfected cell line were stably labeled with fluorescence varying in emission spectra from green to near infrared. After establishing and validating a SHIN3 cell line stably transfected with HaloTagR (HaloTagR-SHIN3), in vivo spectral fluorescence imaging studies were performed in live animals using a peritoneal dissemination model. The tumor nodules arising from HaloTagR-SHIN3 could be successfully labeled by 4 different fluorophore-conjugated HaloTag-ligands each emitting light at different wavelengths. These fluorophores could be alternated on serial imaging sessions permitting assessment of interval growth. Fluorescence was retained in histological specimens after fixation. Thus, this tagging system proves versatile both for in vivo and in vitro imaging without requiring modification of the underlying cell line. Thus, this strategy can overcome some of the limitations associated with the use of endogenous fluorescent proteins and exogenous targeted optical agents in current use. PMID:19514716

  17. Fluorescent naphthalene diols as bridging ligands in Ln(III) cluster chemistry: synthetic, structural, magnetic, and photophysical characterization of Ln(III)8 "Christmas stars".

    PubMed

    Alexandropoulos, Dimitris I; Fournet, Adeline; Cunha-Silva, Luís; Mowson, Andrew M; Bekiari, Vlasoula; Christou, George; Stamatatos, Theocharis C

    2014-06-01

    The initial employment of the fluorescent bridging ligand naphthalene-2,3-diol in 4f-metal coordination chemistry has provided access to a new family of Ln(III)8 clusters with a "Christmas-star" topology, single-molecule magnetism behavior, and ligand-centered emissions. PMID:24828892

  18. Blue fluorescence of three metal-organic zinc polymers containing tetrazinc units and asymmetric ligand of btc{sup 3-}

    SciTech Connect

    Xu Ling; Liu Bing; Zheng Fakun; Guo Guocong . E-mail: gcguo@ms.fjirsm.ac.cn; Huang Jinshun

    2005-11-15

    Three new zinc coordination polymers [Zn{sub 2}(btc){sub 2}(H{sub 2}O){sub 2}] {sub n} .n[Zn(H{sub 2}O){sub 6}] (1), [Zn{sub 3}(btc){sub 2}(2,2'-bipy){sub 2}(H{sub 2}O){sub 3}] {sub n} .2nH{sub 2}O (2) and [Zn{sub 3}(btc){sub 2}(H{sub 2}O){sub 6}] {sub n} .nH{sub 2}O (3) (H{sub 3}btc=1,2,4-benzenetricarboxylic acid, 2,2'-bipy=2,2'-bipyridine) were obtained by the diffusion method and their crystal structures were determined by single-crystal X-ray diffraction. Compounds 1-3 have the similar tetrametallic unit [Zn{sub 4}(btc){sub 2}] SBUs and these SBUs are further connected into stair-like structure, 2-D layer and 3-D framework for 1, 2 and 3, in which the btc{sup 3-} ligands adopt {mu} {sub 3}, {mu} {sub 4} and {mu} {sub 5} coordination modes, respectively. The title compounds show strong blue fluorescence, which may be assigned as {pi}*{sup {yields}}n transition of the ligand mixed with the ligand-to-metal change transfer (LMCT), indicating the fluorescence, indicates the title compounds may be good candidates for blue-light photoactive materials.

  19. Homogeneous time-resolved G protein-coupled receptor-ligand binding assay based on fluorescence cross-correlation spectroscopy.

    PubMed

    Antoine, Thomas; Ott, David; Ebell, Katharina; Hansen, Kerrin; Henry, Luc; Becker, Frank; Hannus, Stefan

    2016-06-01

    G protein-coupled receptors (GPCRs) mediate many important physiological functions and are considered as one of the most successful therapeutic target classes for a wide spectrum of diseases. Drug discovery projects generally benefit from a broad range of experimental approaches for screening compound libraries and for the characterization of binding modes of drug candidates. Owing to the difficulties in solubilizing and purifying GPCRs, assay formats have been so far mainly limited to cell-based functional assays and radioligand binding assays. In this study, we used fluorescence cross-correlation spectroscopy (FCCS) to analyze the interaction of detergent-solubilized receptors to various types of GPCR ligands: endogenous peptides, small molecules, and a large surrogate antagonist represented by a blocking monoclonal antibody. Our work demonstrates the suitability of the homogeneous and time-resolved FCCS assay format for a robust, high-throughput determination of receptor-ligand binding affinities and kinetic rate constants for various therapeutically relevant GPCRs. PMID:26954998

  20. Fluorescence in the system Eu(III) - oxytetracycline - co-ligand -sodium dodecylbenzene sulphonate micelles and its analytical application

    NASA Astrophysics Data System (ADS)

    Shtykov, Sergei N.; Smirnova, Tatyana D.; Kalashnikova, Natalja V.; Bylinkin, Yurii G.; Zhemerichkin, Dmitry A.

    2006-07-01

    Fluorescence enhancement of the Eu 3+ - oxytetracycline (OTC) chelate by addition of phenanthroline (Phen) and trioctyiphosphine oxide (TOPO) as well as micelles of anionic, catiomic and nonionic surfactants has been studied. As was found, in the presence of Phen as co-ligand and micelles of dodecylbenzene sulfonate as anionic surfactant the analytical signal increased by a factor of 8.5 and reached maximum value at pH 8.0 +/- 0.5. The dynamic concentration range of OTC determination was found to be 8.0 x 10 -8 - 4.0 × 10 -5 M (R2 = 0.991) and the detection limit 5.3 × 10 -8 M (3 σ criterion). The procedure based on europium-sensitized fluorescence has been developed for the determination of OTC in chicken meat with the recovery of 98.0-103.3%.

  1. Growth of fluorescence gold clusters using photo-chemically activated ligands

    NASA Astrophysics Data System (ADS)

    Mishra, Dinesh; Aldeek, Fadi; Michael, Serge; Palui, Goutam; Mattoussi, Hedi

    2016-03-01

    Ligands made of lipoic acid (LA) appended with a polyethylene glycol (PEG) chain have been used in the aqueous phase growth of luminescent gold clusters with distinct emission from yellow to near-IR, using two different routes. In the first route, the gold-ligand complex was chemically reduced using sodium borohydride in alkaline medium, which gave near- IR luminescent gold clusters with maximum emission around 745 nm. In the second method, LA-PEG ligand was photochemically modified to a mixture of thiols, oligomers and oxygenated species under UV-irradiation, which was then used as both reducing agent and stabilizing ligand. By adjusting the pH, temperature, and time of the reaction, we were able to obtain clusters with two distinct emission properties. Refluxing the gold-ligand complex in alkaline medium in the presence of excess ligand gave yellow emission within the first two hours and the emission shifted to red after overnight reaction. Mass spectrometry and chemical assay were used to understand the photo-chemical transformation of Lipoic Acid (LA). Mass spectroscopic studies showed the photo-irradiated product contains thiols, oligomers (dimers, trimers and tetramers) as well as oxygenated species. The amount of thiol formed under different conditions of irradiation was estimated using Ellman's assay.

  2. 5f state interaction with inner coordination sphere ligands: einsteinium 3+ ion fluorescence in aqueous and organic phases

    SciTech Connect

    Beitz, J.V.; Wester, D.W.; Williams, C.W.

    1983-01-01

    The interaction between 5f electron states of einsteinium 3+ ion and coordinated ligands in solution has been probed using laser-induced fluorescence. Aquo einsteinium 3+ ion was observed to fluoresce from its first excited J = 5 state in a broad-band peaking at 9260 wavenumbers. The observed fluorescence lifetimes were 1.05 microseconds and 2.78 microseconds in H/sub 2/O and D/sub 2/O (99+ % D atom), respectively. The non-radiative decay rates derived from the lifetime data are compared with previously reported data for Cm, Sm, Eu, Tb, and Dy aquo 3+ ions. The 5f actinide states exhibit substantially greater non-radiative decay rates than do lanthanide 4f states of similar energy gap. This provides evidence that actinide 5f electrons interact more strongly with their inner coordination sphere than do lanthanide ion 4f electrons. The fluorescence lifetime of einsteinium 3+ ion complexed with 1 formal di(2-ethylhexyl)orthophosphoric acid in h-heptane was 2.34 microseconds. 3 figures, 1 table.

  3. Different Ligands of the TRPV3 Cation Channel Cause Distinct Conformational Changes as Revealed by Intrinsic Tryptophan Fluorescence Quenching*

    PubMed Central

    Billen, Bert; Brams, Marijke; Debaveye, Sarah; Remeeva, Alina; Alpizar, Yeranddy A.; Waelkens, Etienne; Kreir, Mohamed; Brüggemann, Andrea; Talavera, Karel; Nilius, Bernd; Voets, Thomas; Ulens, Chris

    2015-01-01

    TRPV3 is a thermosensitive ion channel primarily expressed in epithelial tissues of the skin, nose, and tongue. The channel has been implicated in environmental thermosensation, hyperalgesia in inflamed tissues, skin sensitization, and hair growth. Although transient receptor potential (TRP) channel research has vastly increased our understanding of the physiological mechanisms of nociception and thermosensation, the molecular mechanics of these ion channels are still largely elusive. In order to better comprehend the functional properties and the mechanism of action in TRP channels, high-resolution three-dimensional structures are indispensable, because they will yield the necessary insights into architectural intimacies at the atomic level. However, structural studies of membrane proteins are currently hampered by difficulties in protein purification and in establishing suitable crystallization conditions. In this report, we present a novel protocol for the purification of membrane proteins, which takes advantage of a C-terminal GFP fusion. Using this protocol, we purified human TRPV3. We show that the purified protein is a fully functional ion channel with properties akin to the native channel using planar patch clamp on reconstituted channels and intrinsic tryptophan fluorescence spectroscopy. Using intrinsic tryptophan fluorescence spectroscopy, we reveal clear distinctions in the molecular interaction of different ligands with the channel. Altogether, this study provides powerful tools to broaden our understanding of ligand interaction with TRPV channels, and the availability of purified human TRPV3 opens up perspectives for further structural and functional studies. PMID:25829496

  4. Evaluating the binding efficiency of pheromone binding protein with its natural ligand using molecular docking and fluorescence analysis

    NASA Astrophysics Data System (ADS)

    Ilayaraja, Renganathan; Rajkumar, Ramalingam; Rajesh, Durairaj; Muralidharan, Arumugam Ramachandran; Padmanabhan, Parasuraman; Archunan, Govindaraju

    2014-06-01

    Chemosignals play a crucial role in social and sexual communication among inter- and intra-species. Chemical cues are bound with protein that is present in the pheromones irrespective of sex are commonly called as pheromone binding protein (PBP). In rats, the pheromone compounds are bound with low molecular lipocalin protein α2u-globulin (α2u). We reported farnesol is a natural endogenous ligand (compound) present in rat preputial gland as a bound volatile compound. In the present study, an attempt has been made through computational method to evaluating the binding efficiency of α2u with the natural ligand (farnesol) and standard fluorescent molecule (2-naphthol). The docking analysis revealed that the binding energy of farnesol and 2-naphthol was almost equal and likely to share some binding pocket of protein. Further, to extrapolate the results generated through computational approach, the α2u protein was purified and subjected to fluorescence titration and binding assay. The results showed that the farnesol is replaced by 2-naphthol with high hydrophobicity of TYR120 in binding sites of α2u providing an acceptable dissociation constant indicating the binding efficiency of α2u. The obtained results are in corroboration with the data made through computational approach.

  5. Identification of Pregnane X Receptor Ligands Using Time-Resolved Fluorescence Resonance Energy Transfer and Quantitative High-Throughput Screening

    PubMed Central

    Shukla, Sunita J.; Nguyen, Dac-Trung; MacArthur, Ryan; Simeonov, Anton; Frazee, William J.; Hallis, Tina M.; Marks, Bryan D.; Singh, Upinder; Eliason, Hildegard C.; Printen, John; Austin, Christopher P.; Inglese, James

    2009-01-01

    Abstract The human pregnane X nuclear receptor (PXR) is a xenobiotic-regulated receptor that is activated by a range of diverse chemicals, including antibiotics, antifungals, glucocorticoids, and herbal extracts. PXR has been characterized as an important receptor in the metabolism of xenobiotics due to induction of cytochrome P450 isozymes and activation by a large number of prescribed medications. Developing methodologies that can efficiently detect PXR ligands will be clinically beneficial to avoid potential drug–drug interactions. To facilitate the identification of PXR ligands, a time-resolved fluorescence resonance energy transfer (TR-FRET) assay was miniaturized to a 1,536-well microtiter plate format to employ quantitative high-throughput screening (qHTS). The optimized 1,536-well TR-FRET assay showed Z′-factors of ≥0.5. Seven- to 15-point concentration–response curves (CRCs) were generated for 8,280 compounds using both terbium and fluorescein emission data, resulting in the generation of 241,664 data points. The qHTS method allowed us to retrospectively examine single concentration screening datasets to assess the sensitivity and selectivity of the PXR assay at different compound screening concentrations. Furthermore, nonspecific assay artifacts such as concentration-based quenching of the terbium signal and compound fluorescence were identified through the examination of CRCs for specific emission channels. The CRC information was also used to define chemotypes associated with PXR ligands. This study demonstrates the feasibility of profiling thousands of compounds against PXR using the TR-FRET assay in a high-throughput format. PMID:19505231

  6. Fluorosomes: Fluorescent Virus-Like Nanoparticles that Represent a Convenient Tool to Visualize Receptor-Ligand Interactions

    PubMed Central

    Wojta-Stremayr, Daniela; Pickl, Winfried F.

    2013-01-01

    Viruses are the smallest life forms and parasitize on many eukaryotic organisms, including humans. Consequently, the study of viruses and viral diseases has had an enormous impact on diverse fields of biology and medicine. Due to their often pathogenic properties, viruses have not only had a strong impact on the development of immune cells but also on shaping entire immune mechanisms in their hosts. In order to better characterize virus-specific surface receptors, pathways of virus entry and the mechanisms of virus assembly, diverse methods to visualize virus particles themselves have been developed in the past decades. Apart from characterization of virus-specific mechanisms, fluorescent virus particles also serve as valuable platforms to study receptor-ligand interactions. Along those lines the authors have developed non-infectious virus-like nanoparticles (VNP), which can be decorated with immune receptors of choice and used for probing receptor-ligand interactions, an especially interesting application in the field of basic but also applied immunology research. To be able to better trace receptor-decorated VNP the authors have developed technology to introduce fluorescent proteins into such particles and henceforth termed them fluorosomes (FS). Since VNP are assembled in a simple expression system relying on HEK-293 cells, gene-products of interest can be assembled in a simple and straightforward fashion—one of the reasons why the authors like to call fluorosomes ‘the poor-man's staining tool’. Within this review article an overview on virus particle assembly, chemical and recombinant methods of virus particle labeling and examples on how FS can be applied as sensors to monitor receptor-ligand interactions on leukocytes are given. PMID:23881135

  7. Investigating the Fluorescence Quenching of Doxorubicin in Folic Acid Solutions and its Relation to Ligand-Targeted Nanocarriers.

    PubMed

    Husseini, Ghaleb A; Kanan, Sofian; Al-Sayah, Mohammad

    2016-02-01

    Folic acid (FA) is one of the most utilized moieties in active (ligand) drug delivery. The folate receptor is widely expressed on the surface of several cell lines and tumors; including ovarian, brain, kidney, breast, and lung cancers. During our previous experiments with Doxorubicin (Dox) encapsulated in folate-targeted micelles, we found that flow cytometry underestimated the amount of drug that accu- mulates inside cells. We attributed this effect to the quenching of Dox by FA and herein investigate this phenomenon in an attempt to obtain a correction factor that could be applied to the fluorescence of Dox in the presence of FA. Initially, we examine the effect of pH on the fluorescence spectra of FA, Dox, equimolar solutions of FA and Dox in water, HCI (0.1 M), and NaOH (0.1 M) solutions. We then measure the effect of the gradual increase of FA concentration on the fluorescence intensity of Dox in phosphate-buffered saline (PBS) solutions (pH of 7.4). Using the Stern-Volmer equation, we estimate the association constant of FA-Dox to be K(SV) = 1.5 x 10(4) M(-1). Such an association constant indicates that at the concentrations of FA used in targeted drug delivery systems, a significant concentration of Dox exists as FA-Dox complexes with a quenched fluorescence. Therefore, we conclude that when Dox is used in FA-active drug delivery systems, a correction factor is needed to predict the correct fluorescence intensity of agent in vitro and in vivo. PMID:27433596

  8. Investigating ligand-receptor interactions at bilayer surface using electronic absorption spectroscopy and Fluorescence Resonance Energy Transfer

    PubMed Central

    Dogra, Navneet; Li, Xuelian; Kohli, Punit

    2012-01-01

    We investigate interactions between receptors and ligands at bilayer surface of polydiacetylene (PDA) liposomal nanoparticles using changes in electronic absorption spectroscopy and Fluorescence Resonance Energy Transfer (FRET). We study the effect of mode of linkage (covalent versus non-covalent) between the receptor and liposome bilayer. We also examine the effect of size dependent interactions between liposome and analyte through electronic absorption and FRET responses. Glucose (receptor) molecules were either covalently or non-covalently attached at the bilayer of nanoparticles, and they provided selectivity for molecular interactions between glucose and glycoprotein ligands of E. coli. The receptor-ligand interactions between glucose and ligand on E. Coli surface induced stress on conjugated PDA chain which resulted in changes (blue to red) in the absorption spectrum of PDA. The changes in electronic absorbance also led to changes in FRET efficiency between conjugated PDA chains (acceptor) and fluorophores (Sulphorhodamine-101) (donor) attached to the bilayer surface. Interestingly, we did not find significant differences in UV-Vis and FRET responses for covalently- and non-covalently-bound glucose to liposomes following their interactions with E. Coli. We attributed these results to close proximity of glucose receptor molecules to the liposome bilayer surface such that induced stress were similar in both the cases. We also found that PDA emission from direct excitation mechanism was ~ 2 - 10 times larger than that of FRET based response. These differences in emission signals were attributed to three major reasons: non-specific interactions between E. Coli and liposomes; size differences between analyte and liposomes; and a much higher PDA concentration with respect to sulpho-rhodamine (SR-101). We have proposed a model to explain our experimental observations. Our fundamental studies reported here will help in enhancing our knowledge regarding interactions

  9. Series of dinuclear and tetranuclear lanthanide clusters encapsulated by salen-type and β-diketionate ligands: single-molecule magnet and fluorescence properties.

    PubMed

    Sun, Wen-Bin; Han, Bing-Lu; Lin, Po-Heng; Li, Hong-Feng; Chen, Peng; Tian, Yong-Mei; Murugesu, Muralee; Yan, Peng-Fei

    2013-10-01

    Three dinuclear [Ln2H2OL(1)2(acac)2]·solvent (1, Ln = Gd, solvent = 2CH2Cl2; 2, Ln = Tb, no solvent; 3, Ln = Er, solvent = (C2H5)2O), and two tetranuclear lanthanide clusters [Ln4(μ3-OH)2L(2)2(acac)6]·2(solvent) (4, Ln = Tb, solvent = CH3OH; 5, Ln = Dy, solvent = CH3CN) were characterized in terms of structure, fluorescence and magnetism. The dinuclear lanthanide complexes were constructed by a rigid salen-type ligand H2L(1) = N,N'-bis(salicylidene)-o-phenylenediamine and β-diketonate (acac = acetylacetonate) ligands, while the tetranuclear clusters were formed from the flexible ligand H2L(2) = N,N'-bis(salicylidene)-1,2-ethanediamine. Crystal structure analysis indicates that the rigid ligand favors the double-decker sandwich structure (Ln2L(1)2), in which the two lanthanide ions have different coordination numbers and geometry, while the more flexible ligand (H2L(2)) leads to planar tetranuclear clusters. The relationship between their respective magnetic anisotropy and ligand-field geometries and their fluorescence properties was investigated. The Dy and Tb-containing clusters exhibit typical visible fluorescence properties, and single-molecule magnet behavior is seen in complex 5. PMID:23887476

  10. Synthesis of BODIPY derivatives substituted with various bioconjugatable linker groups: a construction kit for fluorescent labeling of receptor ligands.

    PubMed

    Heisig, Fabian; Gollos, Sabrina; Freudenthal, Sven J; El-Tayeb, Ali; Iqbal, Jamshed; Müller, Christa E

    2014-01-01

    The goal of the present study was to design small, functionalized green-emitting BODIPY dyes, which can readily be coupled to target molecules such as receptor ligands, or even be integrated into their pharmacophores. A simple two-step one-pot procedure starting from 2,4-dimethylpyrrole and ω-bromoalkylcarboxylic acid chlorides was used to obtain new ω-bromoalkyl-substituted BODIPY fluorophores (1a-1f) connected via alkyl spacers of different length to the 8-position of the fluorescent dye. The addition of radical inhibitors reduced the amount of side products. The ω-bromoalkyl-substituted BODIPYs were further converted to introduce various functional groups: iodo-substituted dyes were obtained by Finkelstein reaction in excellent yields; microwave-assisted reaction with methanolic ammonia led to fast and clean conversion to the amino-substituted dyes; a hydroxyl-substituted derivative was prepared by reaction with sodium ethylate, and thiol-substituted BODIPYs were obtained by reaction of 1a-1f with potassium thioacetate followed by alkaline cleavage of the thioesters. Water-soluble derivatives were prepared by introducing sulfonate groups into the 2- and 6-position of the BODIPY core. The synthesized BODIPY derivatives showed high fluorescent yields and appeared to be stable under basic, reducing and oxidative conditions. As a proof of concept, 2-thioadenosine was alkylated with bromoethyl-BODIPY 1b. The resulting fluorescent 2-substituted adenosine derivative 15 displayed selectivity for the A3 adenosine receptor (ARs) over the other AR subtypes, showed agonistic activity, and may thus become a useful tool for studying A3ARs, or a lead structure for further optimization. The new functionalized dyes may be widely used for fluorescent labeling allowing the investigation of biological targets and processes. PMID:24052460

  11. FRET Studies Between CdTe Capped by Small-Molecule Ligands and Fluorescent Protein

    NASA Astrophysics Data System (ADS)

    Zhang, Yue; Zhou, Dejian; He, Junhui

    2014-12-01

    Water-soluble luminescent semiconductor nanocrystals also known as quantum dots (QDs) that have prominent photostability, wide absorption cross sections and tunable narrow emission, have been shown as promising probes in immunoassays. QDs are often used as donors in fluorescence resonance energy transfer (FRET) based sensors using organic dyes or fluorescent proteins as acceptors. Here, the FRET between a QD donor and fluorescent protein acceptors has been studied. The fluorescent protein (FP)mCherry appended with a hexa-histidine-tag could effectively self-assemble onto CdTe to produce small donor-acceptor distances and hence highly efficient FRET (efficiency > 80%) at relatively low FP:CdTe copy numbers (ca.1). Using the Förster dipole-dipole interaction formula, the Förster radius (R0) and respective donor-acceptor distances for the CdTe-FP FRET systems have been calculated. The binding constants (Kd) of the QD-FP systems have also been evaluated by the emission spectra.

  12. Carbon nanodots as ligand exchange probes in Au@C-dot nanobeacons for fluorescent turn-on detection of biothiols.

    PubMed

    Mandani, Sonam; Sharma, Bhagwati; Dey, Deepa; Sarma, Tridib K

    2015-02-01

    Au nanoparticle-carbon dot core-shell (Au@C-dot) nanocomposite was synthesized in aqueous medium at room temperature using the carbon dots as reducing agents themselves. The carbon nanodots also function as an effective stabilizer by forming a thin layer surrounding Au nanoparticles (Au NPs) similar to self-assembled monolayers. Ligand exchange with thiol containing biomolecules resulted in the release of carbon dots from the Au NP surface leading to an enhancement of fluorescence. Simultaneously the agglomeration of Au NPs stimulated by the interaction of biothiols led to changes in the surface plasmon properties of Au NPs. A detailed spectroscopic investigation revealed a combination of static and dynamic quenching being involved in the process. Thus, the Au nanoparticle-carbon dot composite could be used as a dual colorimetric and fluorometric sensor for biothiols ranging from amino acids, peptides, proteins, enzymes etc. with a detection limit of 50 nM. PMID:25520240

  13. Visualizing Microtubule-Dependent Vasopressin Type 2 Receptor Trafficking Using a New High-Affinity Fluorescent Vasopressin Ligand

    PubMed Central

    Chen, Sylvia; Webber, Matthew J.; Vilardaga, Jean-Pierre; Khatri, Ashok; Brown, Dennis; Ausiello, Dennis A.; Lin, Herbert Y.

    2011-01-01

    The vasopressin receptor type 2 (V2R) is the major target of vasopressin (VP) in renal epithelial cells. Although it is known that VP induces V2R internalization, accumulation in the perinuclear area, and degradation, the V2R intracellular trafficking pathways remain elusive. We visualized this process by developing a new fluorescent VP analog tagged by tetramethylrhodamine (TMR)-[Lys-(PEG)2-Suc-TMR8]VP or (VPTMR). This ligand is fully functional as revealed by its high binding affinity toward V2R [(Kd) =157 ± 52 nm] and ability to increase intracellular cAMP 32-fold. VPTMR induced V2R internalization in LLC-PK1 cells expressing either a FLAG-tagged receptor (FLAG-V2R) or V2R C-terminally tagged with green fluorescent protein (GFP) (V2R-GFP). After internalization, VPTMR and V2R-GFP colocalized in the perinuclear area, suggesting that the hormone and receptor traffic along the same pathway. VPTMR and V2R colocalized initially with the early endosome markers EEA1 and Rab5, and later with the recycling and late endosome markers Rab11 and Rab25. Epifluorescence microscopy of LLC-PK1 cells expressing GFP-tagged microtubules (MT) showed that VPTMR-containing vesicles travel along the MT network, and even remain attached to MT during the metaphase and anaphase of mitosis. Colchicine, a MT-depolymerizing agent, abolished perinuclear accumulation of VPTMR, and Western blot analysis showed that VP-induced V2R-GFP degradation is markedly retarded, but not abolished, by colchicine (10 μM). We conclude that the new VPTMR ligand is suitable for dissecting V2R and VP internalization and trafficking in cells, and that V2R trafficking and down-regulation is an MT-dependent mechanism. PMID:21828182

  14. Carbon nanodots as ligand exchange probes in Au@C-dot nanobeacons for fluorescent turn-on detection of biothiols

    NASA Astrophysics Data System (ADS)

    Mandani, Sonam; Sharma, Bhagwati; Dey, Deepa; Sarma, Tridib K.

    2015-01-01

    Au nanoparticle-carbon dot core-shell (Au@C-dot) nanocomposite was synthesized in aqueous medium at room temperature using the carbon dots as reducing agents themselves. The carbon nanodots also function as an effective stabilizer by forming a thin layer surrounding Au nanoparticles (Au NPs) similar to self-assembled monolayers. Ligand exchange with thiol containing biomolecules resulted in the release of carbon dots from the Au NP surface leading to an enhancement of fluorescence. Simultaneously the agglomeration of Au NPs stimulated by the interaction of biothiols led to changes in the surface plasmon properties of Au NPs. A detailed spectroscopic investigation revealed a combination of static and dynamic quenching being involved in the process. Thus, the Au nanoparticle-carbon dot composite could be used as a dual colorimetric and fluorometric sensor for biothiols ranging from amino acids, peptides, proteins, enzymes etc. with a detection limit of 50 nM.Au nanoparticle-carbon dot core-shell (Au@C-dot) nanocomposite was synthesized in aqueous medium at room temperature using the carbon dots as reducing agents themselves. The carbon nanodots also function as an effective stabilizer by forming a thin layer surrounding Au nanoparticles (Au NPs) similar to self-assembled monolayers. Ligand exchange with thiol containing biomolecules resulted in the release of carbon dots from the Au NP surface leading to an enhancement of fluorescence. Simultaneously the agglomeration of Au NPs stimulated by the interaction of biothiols led to changes in the surface plasmon properties of Au NPs. A detailed spectroscopic investigation revealed a combination of static and dynamic quenching being involved in the process. Thus, the Au nanoparticle-carbon dot composite could be used as a dual colorimetric and fluorometric sensor for biothiols ranging from amino acids, peptides, proteins, enzymes etc. with a detection limit of 50 nM. Electronic supplementary information (ESI) available

  15. Fluorescence spectroscopic studies of tyrosine environment and ligand binding of plant calmodulin

    NASA Astrophysics Data System (ADS)

    Sanyal, Gautam; Thompson, Faith; Puett, David

    1990-05-01

    Recent studies in our laboratories have focused on using tyrosine (Tyr) fluorescence of calmodulin (CaM) and tryptophan (Trp) fluorescence of CaM-bound peptdies as intrinsic probes of structure and interactions of this Ca2+ regulatory protein. Plant CaM contains a single Tyr (Tyr.-l38) and vertebrate CaM contains two (Tyr-99 and Tyr-.l38). Neither protein contains Trp. The fluorescence properties of Tyr-138 of wheat-germ CaM is sensitive to conformational changes induced by perturbations such as Ca2+ ligation or depletion, and pH changes. Effects of these perturbations on quantum yield, lifetime and dynamic quenching of Tyr-l38 fluorescence are reported. We have also studied binding of amphiphilic peptides to wheat-germ CaM. A comparison of wheat CaM induced changes in the fluorescence properties of a single Trp of these peptides with those induced by bovine testes CaM indicate general similarities of the peptide binding surfaces of plant and mammalian CaMs. Frequency domain measurements of decay of intensity and anisotropy have suggested some orientational freedom and local motion of the Trp residue of CaM-bound peptide, independent of the overall protein motion, even when the Trp is expected to be buried in the doubly apolar protein-peptide interface. Calmodulin (CaM) is a ubiquitous calcium binding protein which is believed to regulate several different enzymes in diverse cells (Klee et al., 1982). Much of the structural work to date has been carried out on mammalian CaM. However, CaM has also been isolated from plant and invertebrate sources, and a high degree of sequence homology with vertebrate CaM has been found. The amino acid sequence of wheat germ CaM shows eleven substitutions, two insertions and one deletion compared with the 148.-residue bovine brain CaM (Toda et al., 1985). Specific differences with mammalian CaM at two sites make plant CaM attractive for fluorescence spectroscopic studies. These are: (1) The presence of a single tyrosine residue (Tyr

  16. Ligand-centred fluorescence and electronic relaxation cascade at vibrational time scales in transition-metal complexes.

    PubMed

    Messina, Fabrizio; Pomarico, Enrico; Silatani, Mahsa; Baranoff, Etienne; Chergui, Majed

    2015-11-19

    Using femtosecond-resolved photoluminescence up-conversion, we report the observation of the fluorescence of the high-lying ligand-centered (LC) electronic state upon 266 nm excitation of an iridium complex, Ir(ppy)3, with a lifetime of 70 ± 10 fs. It is accompanied by a simultaneous emission of all lower-lying electronic states, except the lowest triplet metal-to-ligand charge-transfer ((3)MLCT) state that shows a rise on the same time scale. Thus, we observe the departure, the intermediate steps, and the arrival of the relaxation cascade spanning ∼1.6 eV from the (1)LC state to the lowest (3)MLCT state, which then yields the long-lived luminescence of the molecule. This represents the first measurement of the total relaxation time over an entire cascade of electronic states in a polyatomic molecule. We find that the relaxation cascade proceeds in ≤10 fs, which is faster than some of the highest-frequency modes of the system. We invoke the participation of the latter modes in conical intersections and their overdamping to low-frequency intramolecular modes. On the basis of literature, we also conclude that this behavior is not specific to transition-metal complexes but also applies to organic molecules. PMID:26509329

  17. Alzheimer’s Disease Diagnosis by Detecting Exogenous Fluorescent Signal of Ligand Bound to Beta Amyloid in the Lens of Human Eye: An Exploratory Study

    PubMed Central

    Kerbage, Charles; Sadowsky, Carl H.; Jennings, Danna; Cagle, Gerald D.; Hartung, Paul D.

    2013-01-01

    We report results of a clinical exploratory human trial involving 10 participants using a combination of a fluorescent ligand and a laser scanning device, SAPPHIRE System, as an aid in the diagnosis of Probable Alzheimer’s disease (AD). To the best of our knowledge, this is the first time that such a technique has been used in vivo of a human lens. The primary goal of the clinical trial, in addition to safety assessment, was to evaluate efficacy of the system. By detecting specific fluorescent signature of ligand bound beta amyloid in the supranucleus (SN) region of the human lens, a twofold differentiation factor between AD patients and Control groups is achieved. Data from our studies indicates that deeper regions of the SN provide the highest measures of ligand bound fluorescence signal from both controls and patients with AD. In addition, we present preclinical studies that were performed to investigate the binding affinity of the ligand to beta amyloid and evaluate the pharmacokinetics of the ligand in rabbit eyes. Further studies are underway involving a larger population for statistical evaluation of the method. PMID:23750151

  18. A Cu2+-selective fluorescent chemosensor based on BODIPY with two pyridine ligands and logic gate

    NASA Astrophysics Data System (ADS)

    Huang, Liuqian; Zhang, Jing; Yu, Xiaoxiu; Ma, Yifan; Huang, Tianjiao; Shen, Xi; Qiu, Huayu; He, Xingxing; Yin, Shouchun

    2015-06-01

    A novel near-infrared fluorescent chemosensor based on BODIPY (Py-1) has been synthesized and characterized. Py-1 displays high selectivity and sensitivity for sensing Cu2+ over other metal ions in acetonitrile. Upon addition of Cu2+ ions, the maximum absorption band of Py-1 in CH3CN displays a red shift from 603 to 608 nm, which results in a visual color change from pink to blue. When Py-1 is excited at 600 nm in the presence of Cu2+, the fluorescent emission intensity of Py-1 at 617 nm is quenched over 86%. Notably, the complex of Py-1-Cu2+ can be restored with the introduction of EDTA or S2-. Consequently, an IMPLICATION logic gate at molecular level operating in fluorescence mode with Cu2+ and S2- as chemical inputs can be constructed. Finally, based on the reversible and reproducible system, a nanoscale sequential memory unit displaying "Writing-Reading-Erasing-Reading" functions can be integrated.

  19. Fluorescence Spectroscopy of tRNA[superscript Phe] Y Base in the Presence of Mg[superscript 2+] and Small Molecule Ligands

    ERIC Educational Resources Information Center

    Kirk, Sarah R.; Silverstein, Todd P.; McFarlane Holman, Karen L.

    2008-01-01

    This laboratory project is one component of a semester-long advanced biochemistry laboratory course that uses several complementary techniques to study tRNA[superscript Phe] conformational changes induced by ligand binding. In this article we describe a set of experiments in which students use fluorescence spectroscopy to study tRNA[superscript…

  20. Design and study of activatable ("OFF/ON") quantum dots (Qdots): ligand selection for Qdot surface modification for controlling Qdot fluorescence quenching and restoration

    NASA Astrophysics Data System (ADS)

    Teblum, Andrew; Basumallick, Srijita; Shah, Rikhav; Mitra, Rajendra N.; Banerjee, Subhash; Santra, Swadeshmukul

    2012-03-01

    We report design and synthesis of a series of activatable "OFF/ON" CdS:Mn/ZnS quantum dot (Qdot) based sensing probes. The Qdot "OFF" state represent the "quenched state" where the Qdot fluorescence is quenched by ligands attached to Qdot surface. Fluorescence quenching is likely due to ligand assisted electron transfer process. Qdot fluorescence is restored when the electron transfer process is stopped. Using this activatable Qdots, we have successfully demonstrated usefulness of these Qdot probes for reliable detection of toxic cadmium ions in solution, selective detection of glutathione and sensitive detection of intracellular cancer drug release event. In this paper, we will discuss a simple but robust method of making water-soluble CdS:Mn/ZnS Qdots at the room-temperature. Two different water-soluble biomolecules, the N-acetyl cysteine (NAC) and the glutathione (GSH) were used as surface coating ligands. This is a singlestep, one-pot synthesis where the Qdot nanocrystals were grown in the presence of the biomolecules. These Qdots were characterized by fluorescence spectroscopy. Stability of the GSH coated Qdots and the NAC coated Qdots were studied by treating with ethylenediaminetetraacetic acid (EDTA, a strong chelating agent for Zn and Cd ions). Our results show that fluorescence properties of Qdots are affected by the type of surface coated ligands. In comparison to the GSH coated Qdots, the NAC coated Qdots show broad but strong emission towards near infra-red region. When treated with EDTA, fluorescence property of the GSH coated Qdot was affected less than the NAC coated Qdots. This preliminary study shows that NAC coated Qdots could potentially be used to develop activatable ("OFF/ON") probes for potential deep-tissue imaging applications. Similarly, the GSH coated Qdots could be applied for probing desired analytes or for bioimaging purposes in environmentally harsh conditions.

  1. Development and utilization of a fluorescence-based receptor-binding assay for the site 5 voltage-sensitive sodium channel ligands brevetoxin and ciguatoxin.

    PubMed

    McCall, Jennifer R; Jacocks, Henry M; Niven, Susan C; Poli, Mark A; Baden, Daniel G; Bourdelais, Andrea J

    2014-01-01

    Brevetoxins are a family of ladder-frame polyether toxins produced during blooms of the marine dinoflagellate Karenia brevis. Consumption of fish exposed to K. brevis blooms can lead to the development of neurotoxic shellfish poisoning. The toxic effects of brevetoxins are due to activation of voltage-sensitive sodium channels (VSSCs) in cell membranes. Binding of toxins has historically been measured using a radioligand competition assay that is fraught with difficulty. In this study, we developed a novel fluorescence-based binding assay for the brevetoxin receptor. Several fluorophores were conjugated to polyether brevetoxin-2 and used as the labeled ligand. Brevetoxin analogs were able to compete for binding with the fluorescent ligands. This assay was qualified against the standard radioligand receptor assay for the brevetoxin receptor. Furthermore, the fluorescence-based assay was used to determine relative concentrations of toxins in raw extracts of K. brevis culture, and to determine ciguatoxin affinity to site 5 of VSSCs. The fluorescence-based assay was quicker, safer, and far less expensive. As such, this assay can be used to replace the current radioligand assay and will be a vital tool for future experiments examining the binding affinity of various ligands for site 5 on sodium channels. PMID:24830141

  2. Fluorescence spectroscopy of soluble E. coli SPase I Δ2-75 reveals conformational changes in response to ligand binding.

    PubMed

    Bhanu, Meera K; Kendall, Debra A

    2014-04-01

    The bacterial Sec pathway is responsible for the translocation of secretory preproteins. During the later stages of transport, the membrane-embedded signal peptidase I (SPase I) cleaves the signal peptide from a preprotein. We used tryptophan fluorescence spectroscopy of a soluble, catalytically active E. coli SPase I Δ2-75 enzyme to study its dynamic conformational changes while in solution and when interacting with lipids and signal peptides. We generated four single Trp SPase I Δ2-75 mutants, W261, W284, W300, and W310. Based on fluorescence quenching experiments, W300 and W310 were found to be more solvent accessible than W261 and W284 in the absence of ligands. W300 and W310 inserted into lipids, consistent with their location at the enzyme's proposed membrane-interface region, while the solvent accessibilities of W261, W284, and W300 were modified in the presence of signal peptide, suggesting propagation of structural changes beyond the active site in response to peptide binding. The signal peptide binding affinity for the enzyme was measured via FRET experiments and the Kd determined to be 4.4 μM. The location of the peptide with respect to the enzyme was also established; this positioning is crucial for the peptide to gain access to the enzyme active site as it emerges from the translocon into the membrane bilayer. These studies reveal enzymatic structural changes required for preprotein proteolysis as it interacts with its two key partners, the signal peptide and membrane phospholipids. PMID:24115229

  3. Photophysical characterization of fluorescent metal nanoclusters synthesized using oligonucleotides, proteins and small molecule ligands

    NASA Astrophysics Data System (ADS)

    Yeh, Hsin-Chih; Sharma, Jaswinder; Yoo, Hyojong; Martinez, Jennifer S.; Werner, James H.

    2010-02-01

    The size transition from bulk conducting metals to insulating nanoparticles and eventually to single atoms passes through the relatively unexplored few-atom nanocluster region. With dimensions close to the Fermi wavelength, these nanoclusters demonstrate molecule-like properties distinct from bulk metals or atoms, such as discrete and size-tunable electronic transitions which lead to photoluminescence. Current research aims to elucidate the fundamental photophysical properties of metal nanoclusters made by different means and based on different encapsulation agents. Here, we report the study of the photophysical properties, including quantum yields, lifetimes, extinction coefficients, blinking dynamics and sizes, of silver and gold nanoclusters synthesized using oligonucleotides, a protein (bovine serum albumin) and a Good's buffer molecule (MES, 2-(N-morpholino) ethanesulfonic acid) as encapsulation agents. We also investigate the change of photoluminescence as a function of temperature. Furthermore, we show that the fluorescent metal clusters can be used as a donor in forming a resonance energy transfer pair with a commercial organic quencher. These new fluorophores have great potential as versatile tools for a broad range of applications in biological and chemical detection.

  4. A fluorescence-based helicase assay: application to the screening of G-quadruplex ligands

    PubMed Central

    Mendoza, Oscar; Gueddouda, Nassima Meriem; Boulé, Jean-Baptiste; Bourdoncle, Anne; Mergny, Jean-Louis

    2015-01-01

    Helicases, enzymes that unwind DNA or RNA structure, are present in the cell nucleus and in the mitochondrion. Although the majority of the helicases unwind DNA or RNA duplexes, some of these proteins are known to resolve unusual structures such as G-quadruplexes (G4) in vitro. G4 may form stable barrier to the progression of molecular motors tracking on DNA. Monitoring G4 unwinding by these enzymes may reveal the mechanisms of the enzymes and provides information about the stability of these structures. In the experiments presented herein, we developed a reliable, inexpensive and rapid fluorescence-based technique to monitor the activity of G4 helicases in real time in a 96-well plate format. This system was used to screen a series of G4 structures and G4 binders for their effect on the Pif1 enzyme, a 5′ to 3′ DNA helicase. This simple assay should be adaptable to analysis of other helicases and G4 structures. PMID:25765657

  5. Fluorescence signaling of hydrogen sulfide in broad pH range using a copper complex based on BINOL-benzimidazole ligands.

    PubMed

    Sun, Mingtai; Yu, Huan; Li, Huihui; Xu, Hongda; Huang, Dejian; Wang, Suhua

    2015-04-20

    A weakly fluorescent complex derived from a binaphthol-benzimidazole ligand was designed and synthesized for hydrogen sulfide at different pH conditions. It was demonstrated that the probe showed the same reactivity to various hydrogen sulfide species in a broad range of pH values to generate highly fluorescent product through a displacement reaction mechanism, whereas the product's fluorescence spectrum exhibited a hypsochromic shift of ∼73 nm (2393 cm(-1)) as pH increased from neutral to basic, which can be used for distinguishing the various species of hydrogen sulfide. This turn-on fluorescence probe was highly selective and sensitive to hydrogen sulfide with a detection limit of 0.11 μM. It was then applied for evaluating the total content of sulfide (including hydrogen sulfide, hydrosulfide, and sulfide) as well as for the visual detection of gaseous H2S in air using a simple test paper strip. PMID:25839192

  6. PolyA-tailed and fluorophore-labeled aptamer-gold nanoparticle conjugate for fluorescence turn-on bioassay using iodide-induced ligand displacement.

    PubMed

    Li, Wei; Dong, Yifan; Wang, Xi; Li, Hui; Xu, Danke

    2015-04-15

    Depending on the strong affinity of polyA sequence to gold (or silver) surface, applicability of polyA-tailed DNA-gold (or silver) nanoparticle conjugates in homogeneous and heterogeneous protein assays was first demonstrated. Interestingly, when using polyA-tailed, fluophore-labeled DNA-AuNP conjugate, it was found that iodide and thiosulfate anions could act as the ligand displacing reagent to detach polyA-tailed DNA strands from AuNP surface and simultaneously activate the AuNP-quenched fluorophores by destroying the polyA-AuNP interaction via a divide-and-conquer strategy. Based on this new discovery, we have developed a novel, cost-effective and sandwich-type fluorescence turn-on aptasensor for highly sensitive and specific thrombin detection, what took advantage of aptamer-conjugated magnetic beads (apt-MBs) for protein capture and separation, and iodide-induced fluorescence recovery of activatable polyA-based AuNP probes through ligand displacement for fluorescence turn-on detection. This proposed aptasensor could detect thrombin specifically with a detection limit as low as 89pM, which was better than or comparable to many existing fluorescent thrombin assays. Importantly, employment of such polyA-based AuNP conjugate not only avoids the use of thiolated oligonucleotides and thiol-containing displacing reagents, but also offers new possibilities for fabricating convenient and cost-effective bioanalytical applications. PMID:25460880

  7. High-contrast fluorescence sensing of aqueous Cu(I) with triaryl-pyrazoline probes: Dissecting the roles of ligand donor strength and excited state proton transfer

    PubMed Central

    Morgan, M. Thomas; Bagchi, Pritha; Fahrni, Christoph J.

    2012-01-01

    Cu(I)-responsive fluorescent probes based on a photoinduced electron transfer (PET) mechanism generally show incomplete fluorescence recovery relative to the intrinsic quantum yield of the fluorescence reporter. Previous studies on probes with an N-aryl thiazacrown Cu(I)-receptor revealed that the recovery is compromised by incomplete Cu(I)-N coordination and resultant ternary complex formation with solvent molecules. Building upon a strategy that successfully increased the fluorescence contrast and quantum yield of Cu(I) probes in methanol, we integrated the arylamine PET donor into the backbone of a hydrophilic thiazacrown ligand with a sulfonated triarylpyrazoline as a water-soluble fluorescence reporter. This approach was not only expected to disfavor ternary complex formation in aqueous solution but also to maximize PET switching through a synergistic Cu(I)-induced conformational change. The resulting water-soluble probe 1 gave a strong 57-fold fluorescence enhancement upon saturation with Cu(I) with high selectivity over other cations, including Cu(II), Hg(II), and Cd(II); however, the recovery quantum yield did not improve over probes with the original N-aryl thiazacrown design. Concluding from detailed photophysical data, including responses to acidification, solvent isotope effects, quantum yields, and time-resolved fluorescence decay profiles, the fluorescence contrast of 1 is compromised by inadequate coordination of Cu(I) to the weakly basic arylamine nitrogen of the PET donor and by fluorescence quenching via two distinct excited state proton transfer pathways operating under neutral and acidic conditions. PMID:23169532

  8. Zn(II)-coordination modulated ligand photophysical processes – the development of fluorescent indicators for imaging biological Zn(II) ions

    PubMed Central

    Yuan, Zhao; Simmons, J. Tyler; Sreenath, Kesavapillai

    2014-01-01

    Molecular photophysics and metal coordination chemistry are the two fundamental pillars that support the development of fluorescent cation indicators. In this article, we describe how Zn(II)-coordination alters various ligand-centered photophysical processes that are pertinent to developing Zn(II) indicators. The main aim is to show how small organic Zn(II) indicators work under the constraints of specific requirements, including Zn(II) detection range, photophysical requirements such as excitation energy and emission color, temporal and spatial resolutions in a heterogeneous intracellular environment, and fluorescence response selectivity between similar cations such as Zn(II) and Cd(II). In the last section, the biological questions that fluorescent Zn(II) indicators help to answer are described, which have been motivating and challenging this field of research. PMID:25071933

  9. Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell.

    PubMed

    Chen, Yunfeng; Liu, Baoyu; Ju, Lining; Hong, Jinsung; Ji, Qinghua; Chen, Wei; Zhu, Cheng

    2015-01-01

    Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions are likely affected by the mechanical environment in which binding and signaling take place. A recent study demonstrated that mechanical force can regulate antigen recognition by and triggering of the T-cell receptor (TCR). This was made possible by a new technology we developed and termed fluorescence biomembrane force probe (fBFP), which combines single-molecule force spectroscopy with fluorescence microscopy. Using an ultra-soft human red blood cell as the sensitive force sensor, a high-speed camera and real-time imaging tracking techniques, the fBFP is of ~1 pN (10(-12) N), ~3 nm and ~0.5 msec in force, spatial and temporal resolution. With the fBFP, one can precisely measure single receptor-ligand binding kinetics under force regulation and simultaneously image binding-triggered intracellular calcium signaling on a single live cell. This new technology can be used to study other membrane receptor-ligand interaction and signaling in other cells under mechanical regulation. PMID:26274371

  10. Four 1-D metal-organic polymers self-assembled from semi-flexible benzimidazole-based ligand: Syntheses, structures and fluorescent properties

    NASA Astrophysics Data System (ADS)

    Zhou, Chun-lin; Wang, Shi-min; Liu, Sai-nan; Yu, Tian-tian; Li, Rui-ying; Xu, Hong; Liu, Zhong-yi; Sun, Huan; Cheng, Jia-jia; Li, Jin-peng; Hou, Hong-wei; Chang, Jun-biao

    2016-08-01

    Four one-dimensional (1-D) metal-organic polymers based on methylene-bis(1,1‧-benzimidazole)(mbbz), namely, {[Hg(mbbz)(SCN)2]·1/3H2O}n (1), [Co(mbbz)(Cl)2]n (2), {[Co(mbbz)(SO4)]·CH3OH}n (3) and {[Zn(mbbz)(SO4)]·CH3OH}n (4) have been successfully synthesized and structurally characterized. Single-crystal X-ray diffraction reveals that polymers 1 and 2 exhibit interesting 1-D double helical chain structures, while polymers 3 and 4 are 1-D double chain structures due to the bridging effect of mbbz ligands and sulfate anions. These polymers containing the mbbz-based ligand have a high degree of dependence on the corresponding counter anions. Furthermore, the fluorescence properties of the four polymers were also investigated in the solid state, showing the fluorescence signal changes in comparing with that of free ligand mbbz.

  11. Water-soluble gold nanoclusters prepared by protein-ligand interaction as fluorescent probe for real-time assay of pyrophosphatase activity.

    PubMed

    Deng, Hao-Hua; Wang, Fei-Fei; Shi, Xiao-Qiong; Peng, Hua-Ping; Liu, Ai-Lin; Xia, Xing-Hua; Chen, Wei

    2016-09-15

    This paper reports a new and facile method for the synthesis of water-soluble thiolate-protected AuNCs via protein-ligand interaction. Using 3-mercaptopropionic acid (MPA) as a model ligand and bovine serum albumin (BSA) as a model protein, water-soluble AuNCs (BSA/MPA-AuNCs) with intense orange-yellow fluorescent emission (quantum yield=16%) are obtained. Results show that AuNCs produced with this method have hydrophobic interactions with BSA. The synthetic strategy is then successfully extended to produce water-soluble AuNCs protected by other thiolates. Moreover, a sensitive and eco-friendly sensing system is established for detection of the activity of inorganic pyrophosphatase (PPase), which relies on the selective coordination of Fe(3+)with BSA/MPA-AuNCs, the higher affinity between pyrophosphate (PPi) and Fe(3+), and the hydrolysis of PPi by PPase. A good linearity between the fluorescence intensity and PPase activity within the range from 0.1 to 3U/L is found, with a detection limit down to 0.07U/L. Additionally, the fluorescent assay developed here is utilized to assay the PPase activity in real biological samples and as well as to evaluate PPase inhibitor, illustrating the great potential for biological analysis. PMID:27093483

  12. Binding-induced fluorescence of serotonin transporter ligands: A spectroscopic and structural study of 4-(4-(dimethylamino)phenyl)-1-methylpyridinium (APP(+)) and APP(+) analogues.

    PubMed

    Wilson, James N; Ladefoged, Lucy Kate; Babinchak, W Michael; Schiøtt, Birgit

    2014-04-16

    The binding-induced fluorescence of 4-(4-(dimethylamino)-phenyl)-1-methylpyridinium (APP(+)) and two new serotonin transporter (SERT)-binding fluorescent analogues, 1-butyl-4-[4-(1-dimethylamino)phenyl]-pyridinium bromide (BPP(+)) and 1-methyl-4-[4-(1-piperidinyl)phenyl]-pyridinium (PPP(+)), has been investigated. Optical spectroscopy reveals that these probes are highly sensitive to their chemical microenvironment, responding to variations in polarity with changes in transition energies and responding to changes in viscosity or rotational freedom with emission enhancements. Molecular docking calculations reveal that the probes are able to access the nonpolar and conformationally restrictive binding pocket of SERT. As a result, the probes exhibit previously not identified binding-induced turn-on emission that is spectroscopically distinct from dyes that have accumulated intracellularly. Thus, binding and transport dynamics of SERT ligands can be resolved both spatially and spectroscopically. PMID:24460204

  13. Binding-Induced Fluorescence of Serotonin Transporter Ligands: A Spectroscopic and Structural Study of 4-(4-(Dimethylamino)phenyl)-1-methylpyridinium (APP+) and APP+ Analogues

    PubMed Central

    2014-01-01

    The binding-induced fluorescence of 4-(4-(dimethylamino)-phenyl)-1-methylpyridinium (APP+) and two new serotonin transporter (SERT)-binding fluorescent analogues, 1-butyl-4-[4-(1-dimethylamino)phenyl]-pyridinium bromide (BPP+) and 1-methyl-4-[4-(1-piperidinyl)phenyl]-pyridinium (PPP+), has been investigated. Optical spectroscopy reveals that these probes are highly sensitive to their chemical microenvironment, responding to variations in polarity with changes in transition energies and responding to changes in viscosity or rotational freedom with emission enhancements. Molecular docking calculations reveal that the probes are able to access the nonpolar and conformationally restrictive binding pocket of SERT. As a result, the probes exhibit previously not identified binding-induced turn-on emission that is spectroscopically distinct from dyes that have accumulated intracellularly. Thus, binding and transport dynamics of SERT ligands can be resolved both spatially and spectroscopically. PMID:24460204

  14. Selective fluorescence sensors for detection of nitroaniline and metal Ions based on ligand-based luminescent metal-organic frameworks

    NASA Astrophysics Data System (ADS)

    Yu, Zongchao; Wang, Fengqin; Lin, Xiangyi; Wang, Chengmiao; Fu, Yiyuan; Wang, Xiaojun; Zhao, Yongnan; Li, Guodong

    2015-12-01

    Metal-organic frameworks (MOFs) are porous crystalline materials with high potential for applications in fluorescence sensors. In this work, two solvent-induced Zn(II)-based metal-organic frameworks, Zn3L3(DMF)2 (1) and Zn3L3(DMA)2(H2O)3 (2) (L=4,4‧-stilbenedicarboxylic acid), were investigated as selective sensing materials for detection of nitroaromatic compounds and metal ions. The sensing experiments show that 1 and 2 both exhibit selective fluorescence quenching toward nitroaniline with a low detection limit. In addition, 1 exhibits high selectivity for detection of Fe3+ and Al3+ by significant fluorescence quenching or enhancement effect. While for 2, it only exhibits significant fluorescence quenching effect for Fe3+. The results indicate that 1 and 2 are both promising fluorescence sensors for detecting and recognizing nitroaniline and metal ions with high sensitivity and selectivity.

  15. Syntheses, structural variations and fluorescence studies of two dinuclear zinc(II) complexes of a Schiff base ligand with an extended carboxylate side arm

    NASA Astrophysics Data System (ADS)

    Shit, Shyamapada; Sasmal, Ashok; Dhal, Piu; Rizzoli, Corrado; Mitra, Samiran

    2016-03-01

    A potentially tetradentate Schiff base ligand containing carboxylic acid group, HL, (E)-2-((pyridin-2-yl)methyleneamino)-5-chlorobenzoic acid is synthesized and characterized. Reaction of HL with hydrated zinc(II) trichloroacetate and zinc(II) trifluoroacetate under similar reaction condition yields two discrete dinuclear complexes, [Zn(L)(Cl)]2 (1) and [Zn(L)(CF3COO)]2 (2) and characterized by different physicochemical methods. Single crystal X-ray structural characterization reveals different ligating properties of the coordinated anionic ligand (L-) in its zinc(II) complexes. The side arm carboxylate of L- shows μ1,3-carboxylato-bridging mode in 1 and connects zinc(II) atoms in syn-anti fashion while it exhibits a μ1,1-carboxylato-bridging mode in 2. The metal ions display distorted square pyramidal geometries in both the structures and associated with different degrees of distortions. The fluorescence spectra of HL and its zinc(II) complexes recorded in methanol at room temperature which reveal the enhancement of emission intensity for the complexes compared to that of the free ligand. Thermogravimetric analyses (TGA) reveal high thermal stabilities of the complexes.

  16. Fluorescence Resonance Energy Transfer Glucose Sensor from Site-Specific Dual Labeling of Glucose/Galactose Binding Protein Using Ligand Protection

    PubMed Central

    Hsieh, Helen V.; Sherman, Douglas B.; Andaluz, Sandra A.; Amiss, Terry J.; Pitner, J. Bruce

    2012-01-01

    Background Site-selective modification of proteins at two separate locations using two different reagents is highly desirable for biosensor applications employing fluorescence resonance energy transfer (FRET), but few strategies are available for such modification. To address this challenge, sequential selective modification of two cysteines in glucose/galactose binding protein (GGBP) was demonstrated using a technique we call “ligand protection.” Method In this technique, two cysteines were introduced in GGBP and one cysteine is rendered inaccessible by the presence of glucose, thus allowing sequential attachment of two different thiol-reactive reagents. The mutant E149C/A213C/L238S was first labeled at E149C in the presence of the ligand glucose. Following dialysis and removal of glucose, the protein was labeled with a second dye, either Texas Red (TR) C5 bromoacetamide or TR C2 maleimide, at the second site, A213C. Results Changes in glucose-dependent fluorescence were observed that were consistent with FRET between the nitrobenzoxadiazole and TR fluorophores. Comparison of models and spectroscopic properties of the C2 and C5 TR FRET constructs suggests the greater rigidity of the C2 linker provides more efficient FRET. Conclusions The ligand protection strategy provides a simple method for labeling GGBP with two different fluorophores to construct FRET-based glucose sensors with glucose affinity within the human physiological glucose range (1–30 mM). This general strategy may also have broad utility for other protein-labeling applications. PMID:23294773

  17. Fluorescent sensing and electrocatalytic properties of three Zn(II)/Co(II) coordination complexes containing two different dicarboxylates and two various bis(pyridyl)-bis(amide) ligands

    NASA Astrophysics Data System (ADS)

    Lin, Hongyan; Rong, Xing; Liu, Guocheng; Wang, Xiang; Wang, Xiuli; Duan, Surui

    2016-09-01

    Three new transition metal(II) coordination complexes constructed from two different dicarboxylates (1,3-H2BDC = 1,3-benzenedicarboxylic acid, 1,4-H2NDC = 1,4-naphthalenedicarboxylic acid) and two bis(pyridyl)-bis(amide) ligands (3-bpcd = N,N‧-bis(3-pyridyl)cyclohexane-1,4-dicarboxamide, 3-bpod = N,N‧-bis(3-pyridyl)octandiamide), [Zn(1,3-BDC)(3-bpcd)0.5(H2O)]·H2O (1), [Zn(1,3-BDC)(3-bpod)0.5(H2O)] (2) and [Co(1,4-NDC)(3-bpod)1.5(H2O)] (3) have been synthesized in the hydrothermal environments and structurally characterized by IR, TG and single crystal X-ray diffraction. Complexes 1 and 2 possess the similar 1D ladder-like chain based on [Zn(1,3-BDC)]n zigzag chain and the bidentate ligands 3-bpcd/or 3-bpod. Complex 3 shows a 2D layered structure with a 5-connected {410} topology, which consists of 1D linear [Co(1,4-NDC)]n chain and [Co(3-bpod)1.5]n chain with alternating arrangement of 3-bpod ligands and Co2(3-bpod)2 dinuclear loops. The adjacent 1D chains for 1-2 or the 2D layers for 3 are further extended into 2D or 3D supramolecular frameworks through the hydrogen bonding interactions. Additionally, the solid state fluorescent properties for the title complexes 1-3, the fluorescent sensing behaviors of complexes 1-2 and the electrochemical behaviour of complex 3 have been investigated.

  18. Dicynamide bridged two new zig-zag 1-D Zn(II) coordination polymers of pyrimidine derived Schiff base ligands: Synthesis, crystal structures and fluorescence studies

    NASA Astrophysics Data System (ADS)

    Konar, Saugata

    2015-07-01

    Two new zigzag 1-D polymeric Zn(II) coordination polymers {[Zn(L1)(μ1,5-dca)](H2O)}n (1), {[Zn(L2)(μ1,5-dca)](ClO4)}n (2) of two potentially tridentate NNO-, NNN-, donor Schiff base ligands [2-(2-(4,6-dimethylpyrimidin-2-yl)hydrazono)methyl)phenol] (L1), [1-(4,6-dimethylpyrimidin-2-yl)-2-(dipyridin-2ylmethylene)hydrazine] (L2) have been synthesized and characterized by elemental analyses, IR and 1H NMR, fluorescence spectroscopy and single crystal X-ray crystallography. The dicyanamide ions act as linkers (μ1,5 mode) in the formation of these coordination polymers. Both the complexes 1 and 2 have same distorted square pyramidal geometry around the Zn(II) centres. The weak forces like π⋯π, Csbnd H⋯π, anion⋯π interactions lead to various supramolecular architectures. Complex 1 shows high chelation enhanced fluorescence compared to that of 2. The fluorescence spectral changes observed high selectivity towards Zn(II) over other metal ions such as Mn(II), Co(II), Ni(II), Cu(II).

  19. Novel dual ligand co-functionalized fluorescent gold nanoclusters as a versatile probe for sensitive analysis of Hg(2+) and oxytetracycline.

    PubMed

    Xu, Shenghao; Li, Xiaolin; Mao, Yaning; Gao, Teng; Feng, Xiuying; Luo, Xiliang

    2016-04-01

    In this work, we present a direct one-step strategy for rapidly preparing dual ligand co-functionalized fluorescent Au nanoclusters (NCs) by using threonine (Thr) and 11-mercaptoundecanoic acid (MUA) as assorted reductants and capping agents in aqueous solution at room temperature. Fluorescence spectra, high-resolution transmission electron microscopy (HRTEM), X-ray photoelectron spectroscopy (XPS), dynamic light scattering (DLS), and infrared (IR) spectroscopy were performed to demonstrate the optical properties and chemical composition of the as-prepared AuNCs. They possess many attractive features such as near-infrared emission (λem = 606 nm), a large Stoke's shift (>300 nm), high colloidal stability (pH, temperature, salt, and time stability), and water dispersibility. Subsequently, the as-prepared AuNCs were used as a versatile probe for "turn off" sensing of Hg(2+) based on aggregation-induced fluorescence quenching and for "turn-on" sensing of oxytetracycline (OTC). This assay provided good linearity ranging from 37.5 to 3750 nM for Hg(2+) and from 0.375 to 12.5 μM for OTC, with detection limits of 8.6 nM and 0.15 μM, respectively. Moreover, the practical application of this assay was further validated by detecting OTC in human serum samples. PMID:26597915

  20. Optimization of time-resolved fluorescence assay for detection of europium-tetraazacyclododecyltetraacetic acid-labeled ligand-receptor interactions.

    PubMed

    De Silva, Channa R; Vagner, Josef; Lynch, Ronald; Gillies, Robert J; Hruby, Victor J

    2010-03-01

    Lanthanide-based luminescent ligand binding assays are superior to traditional radiolabel assays due to improving sensitivity and affordability in high-throughput screening while eliminating the use of radioactivity. Despite significant progress using lanthanide(III)-coordinated chelators such as diethylenetriaminepentaacetic acid (DTPA) derivatives, dissociation-enhanced lanthanide fluoroimmunoassays (DELFIAs) have not yet been successfully used with more stable chelators (e.g., tetraazacyclododecyltetraacetic acid [DOTA] derivatives) due to the incomplete release of lanthanide(III) ions from the complex. Here a modified and optimized DELFIA procedure incorporating an acid treatment protocol is introduced for use with Eu(III)-DOTA-labeled peptides. Complete release of Eu(III) ions from DOTA-labeled ligands was observed using hydrochloric acid (2.0M) prior to the luminescent enhancement step. [Nle(4),d-Phe(7)]-alpha-melanocyte-stimulating hormone (NDP-alpha-MSH) labeled with Eu(III)-DOTA was synthesized, and the binding affinity to cells overexpressing the human melanocortin-4 (hMC4) receptor was evaluated using the modified protocol. Binding data indicate that the Eu(III)-DOTA-linked peptide bound to these cells with an affinity similar to its DTPA analogue. The modified DELFIA procedure was further used to monitor the binding of an Eu(III)-DOTA-labeled heterobivalent peptide to the cells expressing both hMC4 and cholecystokinin-2 (CCK-2) receptors. The modified assay provides superior results and is appropriate for high-throughput screening of ligand libraries. PMID:19852924

  1. Outstanding blue delayed fluorescence and significant processing stability of cuprous complexes with functional pyridine-pyrazolate diimine ligands.

    PubMed

    Zhang, Qing; Chen, Jun; Wu, Xiao-Yuan; Chen, Xu-Lin; Yu, Rongmin; Lu, Can-Zhong

    2015-04-21

    Thermally activated delayed fluorescence (TADF) of five novel cuprous complexes, with quantum yields near 1 in the solid state, has been realized. Meanwhile, these complexes are significantly stable upon processing with small emission spectral variations from the solid to the thin film to the solvent state. PMID:25784048

  2. Establishing the fluorescent amyloid ligand h-FTAA for studying human tissues with systemic and localized amyloid.

    PubMed

    Sjölander, Daniel; Röcken, Christoph; Westermark, Per; Westermark, Gunilla T; Nilsson, K Peter R; Hammarström, Per

    2016-06-01

    Rapid and accurate detection of amyloid deposits in routine surgical pathology settings are of great importance. The use of fluorescence microscopy in combination with appropriate amyloid specific dyes is very promising in this regard. Here we report that a luminescent conjugated oligothiophene, h-FTAA, rapidly and with high sensitivity and selectivity detects amyloid deposits in verified clinical samples from systemic amyloidosis patients with AA, AL and ATTR types; as well as in tissues laden with localized amyloidosis of AANF, AIAPP and ASem1 type. The probe h-FTAA emitted yellow red fluorescence on binding to amyloid deposits, whereas no apparent staining was observed in surrounding tissue. The only functional structure stained with h-FTAA showing the amyloidotypic fluorescence spectrum was Paneth cell granules in intestine. Screening of 114 amyloid containing tissues derived from 107 verified (Congo red birefringence and/or immunohistochemistry) amyloidosis patients revealed complete correlation between h-FTAA and Congo red fluorescence (107/107, 100% sensitivity). The majority of Congo red negative control cases (27 of 32, 85% specificity) were negative with h-FTAA. Small Congo red negative aggregates in kidney, liver, pancreas and duodenum were found by h-FTAA fluorescence in five control patients aged 72-83 years suffering from diverse diseases. The clinical significance of these false-positive lesions is currently not known. Because h-FTAA fluorescence is one magnitude brighter than Congo red and as the staining is performed four magnitudes lower than the concentration of dye, we believe that these inclusions are beyond detection by Congo red. We conclude that h-FTAA is a fluorescent hypersensitive, rapid and powerful tool for identifying amyloid deposits in tissue sections. Use of h-FTAA can be exploited as a rapid complementary technique for accurate detection of amyloid in routine surgical pathology settings. Our results also implicate the potential of

  3. Segmental dynamics of the cytoplasmic domain of erythrocyte band 3 determined by time-resolved fluorescence anisotropy: sensitivity to pH and ligand binding.

    PubMed

    Thevenin, B J; Periasamy, N; Shohet, S B; Verkman, A S

    1994-03-01

    Interactions between the erythrocyte membrane and its skeleton are mediated primarily by binding of cytoskeletal components to a conformationally sensitive structure, the cytoplasmic domain of band 3 (cdb3). To examine the nanosecond segmental motions of cdb3, band 3 was labeled selectively by fluorescein maleimide at Cys-201 near the proposed hinge in cdb3 about which pH-dependent conformational changes occur. Time-resolved anisotropy of labeled cdb3 in isolated form and in stripped erythrocyte membranes was measured by parallel-acquisition frequency-domain microfluorimetry. Samples had a single-component fluorescein lifetime of approximately 4 ns. Multifrequency phase and modulation data (5-200 MHz) fitted well to a segmental motion model containing two correlation times (tau 1c and tau 2c) and two limiting anisotropies (r1infinity and r2infinity). Measurements in protease-cleaved and denatured samples indicated that tau 1c (100-150 ps) corresponded to rapid rotation of bound fluorescein and tau 2c (2-5 ns) corresponded to segmental motion of cdb3. Both motions were hindered as quantified by nonzero r1infinity and r2infinity. The strong pH dependence of segmental motion correlated with that of cdb3 conformation measured by intrinsic tryptophan fluorescence. Significant changes in cdb3 segmental motion occurred upon interactions with the small ligands 2,3-bisphosphoglycerate and calcium and several glycolytic enzymes known to bind to the N terminus of band 3. These time-resolved fluorescence measurements of the nanosecond segmental dynamics of a labeled membrane protein provide evidence for the sensitivity of cdb3 conformation to ligand binding and suggest long-range structural communication through cdb3. PMID:8127875

  4. Functional immobilization of biomembrane fragments on planar waveguides for the investigation of side-directed ligand binding by surface-confined fluorescence.

    PubMed

    Pawlak, M; Grell, E; Schick, E; Anselmetti, D; Ehrat, M

    1998-01-01

    A method for the functional immobilization of Na,K-ATPase-rich membrane fragments on planar metal oxide waveguides has been developed. A novel optical technique based on the highly sensitive detection of surface-confined fluorescence in the evanescent field of the waveguide allowed us to investigate the interactions of the immobilized protein with cations and ligands. For specific binding studies, a FITC-Na,K-ATPase was used, which had been labelled covalently within the ATP-binding domain of the protein. Fluorophore labels of the surface-bound enzyme can be selectively excited in the evanescent field. A preserved functional activity of the immobilized enzyme was only found when a phospholipid monolayer was preassembled onto the hydrophobic chip surface to form a gentle, biocompatible interface. In situ atomic force microscopy (AFM) was used to examine and optimize the conditions for the lipid and membrane fragment assembly and the quality of the formed layers. The enzyme's functional activity was tested by selective K+ cation binding, interaction with anti-fluorescein antibody 4-4-20, phosphorylation of the protein and binding of inhibitory ligand ouabain. The comparison with corresponding fluorescence intensity changes found in bulk solution provides information about the side-directed surface binding of the Na,K-ATPase membrane fragments. The affinity constants of K+ ions to the Na,K-ATPase was the same for the immobilized and the non-immobilized enzyme, providing evidence for the highly native environment on the surface. The method for the functional immobilization of membrane fragments on waveguide surfaces will be the basis for future applications in pharmaceutical research where advanced methods for exploring the molecular mechanisms of membrane receptor targets and drug screening are required. PMID:10822614

  5. Fluorescence study of ligand binding to potato tuber pyrophosphate-dependent phosphofructokinase: evidence for competitive binding between fructose-1,6-bisphosphate and fructose-2,6-bisphosphate.

    PubMed

    Podestá, Florencio E; Plaxton, William C

    2003-06-01

    The intrinsic fluorescence of potato tuber pyrophosphate:fructose-6-phosphate 1-phosphotransferase (PFP) was used as an indicator of conformational changes due to ligand binding. Binding of the substrates and the allosteric activator fructose-2,6-bisphosphate was quantitatively compared to their respective kinetic effects on enzymatic activity. PFP exhibited a relatively high affinity for its isolated substrates, relative to the enzyme's respective K(m) (substrate) values. There are two distinct types of fructose-1,6-bisphosphate interaction with PFP, corresponding to catalytic and activatory binding. Activatory fructose-1,6-bisphosphate binding shares several characteristics with fructose-2,6-bisphosphate binding, indicating that both ligands compete for the same allosteric activator site. Activation by fructose-1,6-bisphosphate or fructose-2,6-bisphosphate was exerted primarily on the forward (glycolytic) reaction by greatly increasing the enzyme's affinity for fructose-6-phosphate. Binding of substrates and effectors to PFP and PFP kinetic properties were markedly influenced by assay pH. Results indicate an increased glycolytic role for PFP during cytosolic acidification that accompanies anoxia stress. PMID:12745260

  6. FRET analysis using sperm-activating peptides tagged with fluorescent proteins reveals that ligand-binding sites exist as clusters.

    PubMed

    Arcos-Hernández, César; Romero, Francisco; Sánchez-Guevara, Yoloxochitl; Beltrán, Carmen; Nishigaki, Takuya

    2016-02-01

    Long-range cellular communication between the sperm and egg is critical for external fertilization. Sperm-activating peptides (SAPs) are diffusible components of the outer layer of eggs in echinoderms, and function as chemoattractants for spermatozoa. The decapeptide named speract is the best-characterized sea urchin SAP. Biochemical and physiological actions of speract have been studied with purified or chemically synthesized peptides. In this work, we prepared recombinant speract fused to a fluorescent protein (FP; FP-speract) using three color variants: a cyan (eCFP), a yellow (mVenus) and a large Stokes shift yellow (mAmetrine) FP. Although these fluorescence tags are 20 times larger than speract, competitive binding experiments using mAmetrine-speract revealed that this FP-speract has binding affinity to the receptor that is comparable (7.6-fold less) to that of non-labeled speract. Indeed, 10 nmol l(-1) eCFP-speract induces physiological sperm responses such as membrane potential changes and increases in intracellular pH and Ca(2+) concentrations similar to those triggered by 10 nmol l(-1) speract. Furthermore, FP-speract maintains its fluorescence upon binding to its receptor. Using this property, we performed fluorescence resonance energy transfer (FRET) measurements with eCFP-speract and mVenus-speract as probes and obtained a positive FRET signal upon binding to the receptor, which suggests that the speract receptor exists as an oligomer, at least as a dimer, or alternatively that a single speract receptor protein possesses multiple binding sites. This property could partially account for the positive and/or negative cooperative binding of speract to the receptor. PMID:26889001

  7. A Cu²⁺-selective fluorescent chemosensor based on BODIPY with two pyridine ligands and logic gate.

    PubMed

    Huang, Liuqian; Zhang, Jing; Yu, Xiaoxiu; Ma, Yifan; Huang, Tianjiao; Shen, Xi; Qiu, Huayu; He, Xingxing; Yin, Shouchun

    2015-06-15

    A novel near-infrared fluorescent chemosensor based on BODIPY (Py-1) has been synthesized and characterized. Py-1 displays high selectivity and sensitivity for sensing Cu(2+) over other metal ions in acetonitrile. Upon addition of Cu(2+) ions, the maximum absorption band of Py-1 in CH3CN displays a red shift from 603 to 608 nm, which results in a visual color change from pink to blue. When Py-1 is excited at 600 nm in the presence of Cu(2+), the fluorescent emission intensity of Py-1 at 617 nm is quenched over 86%. Notably, the complex of Py-1-Cu(2+) can be restored with the introduction of EDTA or S(2-). Consequently, an IMPLICATION logic gate at molecular level operating in fluorescence mode with Cu(2+) and S(2-) as chemical inputs can be constructed. Finally, based on the reversible and reproducible system, a nanoscale sequential memory unit displaying "Writing-Reading-Erasing-Reading" functions can be integrated. PMID:25766475

  8. Four Zn(II)/Cd(II)-3-amino-1,2,4-triazolate frameworks constructed by in situ metal/ligand reactions: Structures and fluorescent properties

    SciTech Connect

    Chen Zilu; Li Xiaoling; Liang Fupei

    2008-08-15

    Four Cd(II) and Zn(II) complexes with the in situ-generated ligand of 3-amino-1,2,4-triazolate (AmTAZ{sup -}) were isolated from the solvothermal reactions of the corresponding Cd(II) or Zn(II) salts with 5-amino-1H-1,2,4-triazole-3-carboxylic acid (AmTAZAc). Their structures were determined by single-crystal X-ray diffraction analysis. [Zn(AmTAZ)(CH{sub 3}COO)] (1) presents a two-dimensional framework constructed from Zn(II) ions and {mu}{sub 3}-AmTAZ{sup -} ligands. A remarkable feature of [Zn{sub 4}(AmTAZ){sub 4}(SO{sub 4})(OH)(C{sub 2}O{sub 4}){sub 0.5}].2H{sub 2}O (2) is the construction of the building units of octagonal cylinders which interact with each other by sharing one face or overlapping, resulting in the formation of a three-dimensional framework with three kinds of 1D channels. [Cd(AmTAZ)Br] (3) crystallizes in a chiral space group P2{sub 1}2{sub 1}2{sub 1}, giving a homochiral three-dimensional framework with two types of helical channels (left- and right-handed). Different from the others, the 3-amino-1,2,4-triazole molecules in [Cd(AmTAZH)SO{sub 4}] (4) behave as neutral {mu}{sub 2}-2,4-bridges to connect the two-dimensional CdSO{sub 4} sheets into a three-dimensional framework. Of all, 2 and 3 display different fluorescent properties probably due to different metal ions, coordination environments and structural topologies. - Graphical abstract: The solvothermal reactions of Cd(II) and Zn(II) salts bearing different anions with 5-amino-1H-1,2,4-triazole-3-carboxylic acid (AmTAZAc) produced four Cd(II) and Zn(II) MOFs with the in situ-generated 3-amino-1,2,4-triazolate (AmTAZ{sup -}) ion as ligand, which display different structural topologies and fluorescent properties. Display Omitted.

  9. Coordination polymers of Fe(iii) and Al(iii) ions with TCA ligand: distinctive fluorescence, CO2 uptake, redox-activity and oxygen evolution reaction.

    PubMed

    Dhara, Barun; Sappati, Subrahmanyam; Singh, Santosh K; Kurungot, Sreekumar; Ghosh, Prasenjit; Ballav, Nirmalya

    2016-04-28

    Fe and Al belong to different groups in the periodic table, one from the p-block and the other from the d-block. In spite of their different groups, they have the similarity of exhibiting a stable 3+ oxidation state. Here we have prepared Fe(iii) and Al(iii) based coordination polymers in the form of metal-organic gels with the 4,4',4''-tricarboxyltriphenylamine (TCA) ligand, namely Fe-TCA and Al-TCA, and evaluated some important physicochemical properties. Specifically, the electrical conductivity, redox-activity, porosity, and electrocatalytic activity (oxygen evolution reaction) of the Fe-TCA system were noted to be remarkably higher than those of the Al-TCA system. As for the photophysical properties, almost complete quenching of the fluorescence originating from TCA was observed in case of the Fe-TCA system, whereas for the Al-TCA system a significant retention of fluorescence with red-shifted emission was observed. Quantum mechanical calculations based on density functional theory (DFT) were performed to unravel the origin of such discriminative behaviour of these coordination polymer systems. PMID:26961352

  10. Ligand Based Dual Fluorescence and Phosphorescence Emission from BODIPY Platinum Complexes and Its Application to Ratiometric Singlet Oxygen Detection.

    PubMed

    Geist, Fabian; Jackel, Andrej; Winter, Rainer F

    2015-11-16

    Four new 4,4-difluoro-4-bora-3a,4a-diaza-s-indacen-8-yl (BODIPY) platinum(II) complexes of the type cis-/trans-Pt(BODIPY)Br(PR3)2 (R = Et or Ph) were synthesized and characterized by NMR, electronic absorption, and luminescence spectroscopy. Three of the complexes were also studied by single crystal X-ray diffraction. The absorption profiles of the four complexes feature intense HOMO → LUMO π → π* transitions with molar extinction coefficients ε of ca. 50 000 M(-1)cm(-1) at around 475 nm and vibrational progressions that are characteristic of BODIPYs. Most remarkably, most complexes exhibit dual emissions through fluorescence at ca. 490 nm and phosphorescence at ca. 650 nm that originate from Pt-perturbed BODIPY-centered (1)ππ* or (3)ππ* states, respectively. Electronic absorption and luminescence spectroscopy data are in good agreement with our TD-DFT calculations. While the emission of the cis-complexes is dominated by fluorescence, their trans-isomers emit predominantly through phosphorescence with a phosphorescence quantum yield for trans-Pt(BODIPY)Br(PEt3)2 (trans-1) of 31.2%. trans-1 allows for ratiometric one-component oxygen sensing in fluid solution up to atmospheric concentration levels and exhibits a remarkably high Stern-Volmer constant for the quenching of the excited triplet state by oxygen of ca. 350 bar(-1) as determined by changes in phosphorescence intensity and lifetime. PMID:26540413

  11. Fluorescence properties and sequestration of peripheral anionic site specific ligands in bile acid hosts: Effect on acetylcholinesterase inhibition activity.

    PubMed

    Islam, Mullah Muhaiminul; Aguan, Kripamoy; Mitra, Sivaprasad

    2016-05-01

    The increase in fluorescence intensity of model acetyl cholinesterase (AChE) inhibitors like propidium iodide (PI) and ethidium bromide (EB) is due to sequestration of the probes in primary micellar aggregates of bile acid (BA) host medium with moderate binding affinity of ca. 10(2)-10(3)M(-1). Multiple regression analysis of solvent dependent fluorescence behavior of PI indicates the decrease in total nonradiative decay rate due to partial shielding of the probe from hydrogen bond donation ability of the aqueous medium in bile acid bound fraction. Both PI and EB affects AChE activity through mixed inhibition and consistent with one site binding model; however, PI (IC50=20±1μM) shows greater inhibition in comparison with EB (IC50=40±3μM) possibly due to stronger interaction with enzyme active site. The potency of AChE inhibition for both the compounds is drastically reduced in the presence of bile acid due to the formation of BA-inhibitor complex and subsequent reduction of active inhibitor fraction in the medium. Although the inhibition mechanism still remains the same, the course of catalytic reaction critically depends on equilibrium binding among several species present in the solution; particularly at low inhibitor concentration. All the kinetic parameters for enzyme inhibition reaction are nicely correlated with the association constant for BA-inhibitor complex formation. PMID:26974580

  12. Fluorescence energy transfer measurement of distances between ligand binding sites of tubulin and its implication for protein-protein interaction.

    PubMed Central

    Bhattacharya, A.; Bhattacharyya, B.; Roy, S.

    1996-01-01

    9-(Dicyanovinyl) julolidine (DCVJ) is a fluorescent probe, which binds to a unique site on the tubulin dimer and exhibits different properties that are dependent upon its oligomeric state (Kung & Reed, 1989). DCVJ binds to tubulin, the tubulin-colchicine complex, and the tubulin-ruthenium red complex equally well, but binds tighter to the ANS-tubulin complex than to tubulin alone. The energy transfer studies indicate a small amount of energy transfer with colchicine, but a significant energy transfer with ANS. It was shown previously that ruthenium red binds near the C-terminal tail region of the alpha-subunit. Ruthenium red causes major quenching of fluorescence of the tubulin-DCVJ complex, suggesting proximity of binding sites. The derived distances are consistent with DCVJ binding near the alpha beta interface, but on the opposite face of the colchicine binding site. Location of the binding site correlates with the observed effect of a different polymerized state of tubulin on the DCVJ spectroscopic properties. The effect of dimer-dimer association on DCVJ binding, at high protein concentrations (Kung & Reed, 1989), suggests that such an association may occur through lateral contacts of the elongated tubulin dimer, at least in a significant fraction of the cases. Transmission of ANS-induced conformational change to the DCVJ binding site, which is near important dimer-dimer contact sites, makes it possible that such conformational changes may be responsible for polymerization inhibition by anilino-naphthalene sulfonates. PMID:8897603

  13. Exploring the effect of chain length of bridging ligands in cobalt(II) coordination polymers based on flexible bis(5,6-dimethylbenzimidazole) ligands: Synthesis, crystal structures, fluorescence and catalytic properties

    NASA Astrophysics Data System (ADS)

    Qin, Li; Li, Yue-Hua; Ma, Pei-Juan; Cui, Guang-Hua

    2013-11-01

    Two Co(II) coordination polymers derived from a dicarboxylate and two flexible bis(5,6-dimethylbenzimidazole) ligands with varying chain lengths equipped, namely [Co(bdmbmm)(nip)]n (1) and [Co2(bdmbmb)2(nip)2ṡH2O]n (2) (bdmbmm = 1,1'-bis(5,6-dimethylbenzimidazole)methane, H2nip = 5-nitroisophthalic acid, bdmbmb = 1,4-bis(5,6-dimethylbenzimidazole)butane), have been synthesized by hydrothermal methods and characterized by elemental analyses, IR spectra, thermogravimetric analysis (TGA), X-ray powder diffraction (XRPD) and single-crystal X-ray diffraction. Complex 1 forms a 1D looped-like chain consisting of two kinds of macrocycles, which is further arranged into a 2D supramolecular layer through face-to-face π-π stacking interactions; whereas complex 2 exhibits a 3D framework with a twofold interpenetrating diamondoid topology. The fluorescence and catalytic properties of the complexes for the degradation of methyl orange by sodium persulfate have been investigated.

  14. Binding of polarity-sensitive hydrophobic ligands to erythroid and nonerythroid spectrin: fluorescence and molecular modeling studies.

    PubMed

    Patra, Malay; Mitra, Madhurima; Chakrabarti, Abhijit; Mukhopadhyay, Chaitali

    2014-01-01

    We have used three polarity-sensitive fluorescence probes, 6-propionyl 2-(N,N-dimethyl-amino) naphthalene (Prodan), pyrene and 8-anilino 1-naphthalene sulphonic acid, to study their binding with erythroid and nonerythroid spectrin, using fluorescence spectroscopy. We have found that both bind to prodan and pyrene with high affinities with apparent dissociation constants (Kd) of .50 and .17 μM, for prodan, and .04 and .02 μM, for pyrene, respectively. The most striking aspect of these bindings have been that the binding stoichiometry have been equal to 1 in erythroid spectrin, both in dimeric and tetrameric form, and in tetrameric nonerythroid spectrin. From an estimate of apparent dielectric constants, the polarity of the binding site in both erythroid and nonerythroid forms have been found to be extremely hydrophobic. Thermodynamic parameters associated with such binding revealed that the binding is favored by positive change in entropy. Molecular docking studies alone indicate that both prodan and pyrene bind to the four major structural domains, following the order in the strength of binding to the Ankyrin binding domain > SH3 domain > Self-association domain > N-terminal domain of α-spectrin of both forms of spectrin. The binding experiments, particularly with the tetrameric nonerythroid spectrin, however, indicate more toward the self association domain in offering the unique binding site, since the binding stoichiometry have been 1 in all forms of dimeric and tetrameric spectrin, so far studied by us. Further studies are needed to characterize the hydrophobic binding sites in both forms of spectrin. PMID:24404769

  15. Site-directed tryptophan fluorescence reveals the solution structure of tear lipocalin: evidence for features that confer promiscuity in ligand binding.

    PubMed

    Gasymov, O K; Abduragimov, A R; Yusifov, T N; Glasgow, B J

    2001-12-11

    The solution structure of human TL was deduced from the position of the emission peaks after site-directed tryptophan fluorescence (SDTF). The fluorescent amino acid tryptophan was sequentially substituted for each native amino acid in the sequence. Characteristic periodicities for eight beta-strands that comprise the beta-barrel and three alpha-helices were identified. The putative beta-strand I was relatively exposed to solvent, suggesting it does not participate in the formation of the beta-barrel. The beta-strands A and F contain beta-bulges. The average lambda(max) of emission maxima reveals that strand D is at the edge of the barrel and beta-strand H interacts with the main alpha-helical domain. On the basis of the SDTF data, a 3D homology model was constructed for TL and compared to the known crystallographic structures of RBP and beta-lactoglobulin. The small size and splayed open configuration of the E-F hairpin facilitate access of ligands into the cavity mouth of TL as compared to that of RBP with a long overhanging loop that restricts access. In the model of TL, four alanine residues are positioned in the binding site as compared to bulkier residues in the corresponding positions of beta-lactoglobulin. Substitution of A51, A66, A86 to Trp results in a 3-4-fold decrease in binding affinity. The data suggest that the smaller side chains of Ala provide more capacity in the cavity of TL than the bulkier side chains (I56, I71, V92) in the cavity of beta-lactoglobulin. The structural features provide an explanation for the promiscuous binding characteristics exhibited by TL. SDTF provides a general approach for determining the solution structure of many proteins and enhances homology modeling in the absence of high sequence identity. PMID:11732894

  16. Synthesis of a new N-substituted bis-benzimidazolyl diamide ligand and its trinuclear copper(II) complex: Structural and fluorescence studies

    NASA Astrophysics Data System (ADS)

    Mahiya, Kuldeep; Mathur, Pavan

    2013-09-01

    The synthesis of a new N-substituted fluorescent probe based on a bis-benzimidazole diamide N2,N2‧-bis[(1-(4-methylbenzyl)-benzimidazol-2-yl)methyl]biphenyl-2,2‧-dicarboxamide (L1) with a biphenyl spacer group and its trinuclear copper(II) complex [Cu3(L1)3Cl3]ṡ3Clṡ3H2O] has been described. X-ray studies shows that the trinuclear complex crystallizes as [{Cu3(L1)3Cl3}2ṡ6Clṡ13CH3CNṡ2H2O] in triclinic space group P-1 with two independent molecules in the asymmetric unit. Each copper(II) adopts a distorted penta-coordinated geometry in each unit. The fluorescence spectra of L1 in methanol show an emission band centered at 300 nm. This band arises due to benzimidazolyl moiety in the ligating system. The diamide L1 in the presence of Fe3+ show the simultaneous ‘quenching' of (300 nm) and ‘enhancement' of (375 nm) emission band. The new emission band at 375 nm is attributed to intra ligand π-π* transition of the biphenyl moiety. While Cu2+ and Ag+ show only the quenching of the 300 nm band. No such behavior was observed with other metal ions like Ni2+, Co2+, Mn2+, Mg2+, Zn2+ and Pb2+. The quenching constant with Fe3+, Ag+ and Cu2+ are calculated by the Stern-Volmer plots.

  17. Application of three-coordinate copper(I) complexes with halide ligands in organic light-emitting diodes that exhibit delayed fluorescence.

    PubMed

    Osawa, Masahisa; Hoshino, Mikio; Hashimoto, Masashi; Kawata, Isao; Igawa, Satoshi; Yashima, Masataka

    2015-05-14

    A series of three-coordinate copper(I) complexes (L(Me))CuX [X = Cl (1), Br (2), I (3)], (L(Et))CuBr (4), and (L(iPr))CuBr (5) [L(Me) = 1,2-bis[bis(2-methylphenyl)phosphino]benzene, L(Et) = 1,2-bis[bis(2-ethylphenyl)phosphino]benzene, and L(iPr) = 1,2-bis[bis(2-isopropylphenyl)phosphino]benzene] exhibit efficient blue-green emission in the solid state at ambient temperature with peak wavelengths between 473 and 517 nm. The emission quantum yields were 0.38-0.95. The emission lifetimes were measured in the temperature range of 77-295 K using a nanosecond laser technique. The temperature dependence of the emission lifetimes was explained using a model with two excited states: a singlet and a triplet state. The small energy gaps (<830 cm(-1)) between the two states suggest that efficient emission from 1-5 was thermally activated delayed fluorescence (TADF). Alkyl substituents at ortho positions of peripheral phenyl groups were found to have little effect on the electronic excited states. Because the origin of the emission of complexes 2, 4, and 5 was thought to be a (σ + Br)→π* transition, photoluminescence characteristics of these complexes were dominated by the diphosphine ligands. Complexes 2, 4, and 5 had similar emission properties. Complexes 1-5 had efficient green TADF in amorphous films at 293 K with maximum emission wavelengths of 508-520 nm and quantum yields of 0.61-0.71. Organic light-emitting devices that contained complexes 1-5 and exhibited TADF exhibit bright green luminescence with current efficiencies of 55.6-69.4 cd A(-1) and maximum external quantum efficiencies of 18.6-22.5%. PMID:25470470

  18. Synthesis and characterization of a copper(II) complex of a ONN donor Schiff base ligand derived from pyridoxal and 2-(pyrid-2-yl)ethylamine - A novel pyridoxal based fluorescent probe

    NASA Astrophysics Data System (ADS)

    Mandal, Senjuti; Modak, Ritwik; Goswami, Sanchita

    2013-04-01

    The title complex, Cu(LH)Cl2 is the first copper(II) complex with a Schiff base derived from pyridoxal and 2-(pyrid-2-yl)ethylamine. The central metal lies in a distorted square pyramidal environment with basal plane occupied by the tridentate ONN donor ligand and a Cl atom. The apical position is occupied by another Cl atom. The existence of two different kinds of H-bonds stabilize the network that propagates as parallel layers along crystallographic b axis. The compound exhibits an irreversible CuII/CuI couple in DMF. As pyridoxal containing moieties are fluorescent in nature, its potential as a fluorescent probe is cultivated. Copper(II) ion effectively quenches the fluorescence of HL and the association constant for Cu(II) was estimated to be 10.8 × 104 M-1 in methanol by the linear Benesi-Hildebrand equation.

  19. Synthesis, crystal structure, fluorescence and electrochemical studies of a new tridentate Schiff base ligand and its nickel(II) and palladium(II) complexes

    NASA Astrophysics Data System (ADS)

    Shafaatian, Bita; Soleymanpour, Ahmad; Kholghi Oskouei, Nasim; Notash, Behrouz; Rezvani, Seyyed Ahmad

    2014-07-01

    A new unsymmetrical tridentate Schiff base ligand was derived from the 1:1 M condensation of ortho-vanillin with 2-mercaptoethylamine. Nickel and palladium complexes were obtained by the reaction of the tridentate Schiff base ligand with nickel(II) acetate tetrahydrate and palladium(II) acetate in 2:1 M ratio. In nickel and palladium complexes the ligand was coordinated to metals via the imine N and enolic O atoms. The S groups of Schiff bases were not coordinated to the metals and S-S coupling was occured. The complexes have been found to possess 1:2 Metal:Ligand stoichiometry and the molar conductance data revealed that the metal complexes were non-electrolytes. The complexes exhibited octahedral coordination geometry. The emission spectra of the ligand and its complexes were studied in methanol. Electrochemical properties of the ligand and its metal complexes were investigated in the CH3CN solvent at the 100 mV s-1 scan rate. The ligand and metal complexes showed both reversible and quasi-reversible processes at this scan rate. The Schiff base and its complexes have been characterized by IR, 1H NMR, UV/Vis, elemental analyses and conductometry. The crystal structure of nickel complex has been determined by single crystal X-ray diffraction.

  20. Synthesis, structure, DFT calculations, electrochemistry, fluorescence, DNA binding and molecular docking aspects of a novel oxime based ligand and its palladium(II) complex.

    PubMed

    Bandyopadhyay, Nirmalya; Pradhan, Ankur Bikash; Das, Suman; Lu, Liping; Zhu, Miaoli; Chowdhury, Shubhamoy; Naskar, Jnan Prakash

    2016-07-01

    A novel oxime based ligand, phenyl-(pyridine-2-yl-hydrazono)-acetaldehyde oxime (LH), and its palladium(II) complex (1) have been synthesised and spectroscopically characterised. The ligand crystallizes in the monoclinic space group (P21/c). The X-ray crystal structure of the ligand shows that it forms a hydrogen bonded helical network. The ligand has been characterised by C, H and N microanalyses, (1)H and (13)C NMR, ESI-MS, FT-IR and UV-Vis spectral measurements. Geometry optimizations at the level of DFT show that the Pd(II) centre is nested in a square-planar 'N3Cl' coordination chromophore. The diamagnetic palladium complex has been characterised by C, H and N microanalyses, FAB-MS, FT-IR, UV-Vis spectra and molar electrical conductivity measurements. The observed electronic spectrum of 1 correlates with our theoretical findings as evaluated through TD-DFT. 1 displays quasi-reversible Pd(II)/Pd(III) and Pd(III)/Pd(IV) redox couples in its CV in acetonitrile. 1 is nine-fold more emissive with respect to the binding ligand. Biophysical studies have been carried out to show the DNA binding aspects of both the ligand and complex. The binding constants for the ligand and complex were found to be 3.93×10(4) and 1.38×10(3)M(-1) respectively. To have an insight into the mode of binding of LH and 1 with CT DNA a hydrodynamic study was also undertaken. The mode of binding has also been substantiated through molecular docking. A promising groove binding efficacy has been revealed for the ligand. PMID:27179300

  1. Synthesis, characterization, single crystal X-ray determination, fluorescence and electrochemical studies of new dinuclear nickel(II) and oxovanadium(IV) complexes containing double Schiff base ligands.

    PubMed

    Shafaatian, Bita; Ozbakzaei, Zahra; Notash, Behrouz; Rezvani, S Ahmad

    2015-04-01

    A series of new bimetallic complexes of nickel(II) and vanadium(IV) have been synthesized by the reaction of the new double bidentate Schiff base ligands with nickel acetate and vanadyl acetylacetonate in 1:1 M ratio. In nickel and also vanadyl complexes the ligands were coordinated to the metals via the imine N and enolic O atoms. The complexes have been found to possess 1:1 metals to ligands stoichiometry and the molar conductance data revealed that the metal complexes were non-electrolytes. The nickel and vanadyl complexes exhibited distorted square planar and square pyramidal coordination geometries, respectively. The emission spectra of the ligands and their complexes were studied in methanol. Electrochemical properties of the ligands and their metal complexes were also investigated in DMSO solvent at 150 mV s(-1) scan rate. The ligands and metal complexes showed both quasi-reversible and irreversible processes at this scan rate. The Schiff bases and their complexes have been characterized by FT-IR, 1H NMR, UV/Vis spectroscopies, elemental analysis and conductometry. The crystal structure of the nickel complex has been determined by single crystal X-ray diffraction. PMID:25613693

  2. Palladium(II) and zinc(II) complexes of neutral [N2O2] donor Schiff bases derived from furfuraldehyde: synthesis, characterization, fluorescence and corrosion inhibitors of ligands.

    PubMed

    Ali, Omyma A M

    2014-11-11

    Metal complexes of Schiff bases derived from furfuraldehyde and 4,5-dimethyl-1,2-phenylendiamine (L1) or 4,5-dichloro-1,2-phenylendiamine (L2) have been reported and characterized based on elemental analyses, IR, 1H NMR, UV-Vis, magnetic moment, molar conductance and thermal analysis. The complexes are found to have the formulae [PdL1-2]Cl2 and [ZnL1-2](AcO)2·H2O. The molar conductance data reveal that Pd(II) and Zn(II) chelates are ionic in nature and are of the type 2:1 electrolytes. The spectral data are consistent with a square planar and tetrahedral geometry around Pd(II) and Zn(II), respectively, in which the ligands act as tetradentate ligands. The thermal behavior of some chelates is studied and the activation thermodynamic parameters are calculated using Coats-Redfern method. The corrosion inhibition of stainless steel types 410 and 304 in 1 M HCl using the synthesized Schiff bases as inhibitors have been studied by weight loss method. The obtained data considered these ligands as efficient corrosion inhibitors. The ligands and their metal complexes exhibited considerable antibacterial activity against Staphylococcusaureus, and Escherichiacoli and antifungal activity against Candida albicans. PMID:24858346

  3. Palladium(II) and zinc(II) complexes of neutral [N2O2] donor Schiff bases derived from furfuraldehyde: Synthesis, characterization, fluorescence and corrosion inhibitors of ligands

    NASA Astrophysics Data System (ADS)

    Ali, Omyma A. M.

    2014-11-01

    Metal complexes of Schiff bases derived from furfuraldehyde and 4,5-dimethyl-1,2-phenylendiamine (L1) or 4,5-dichloro-1,2-phenylendiamine (L2) have been reported and characterized based on elemental analyses, IR, 1H NMR, UV-Vis, magnetic moment, molar conductance and thermal analysis. The complexes are found to have the formulae [PdL1-2]Cl2 and [ZnL1-2](AcO)2·H2O. The molar conductance data reveal that Pd(II) and Zn(II) chelates are ionic in nature and are of the type 2:1 electrolytes. The spectral data are consistent with a square planar and tetrahedral geometry around Pd(II) and Zn(II), respectively, in which the ligands act as tetradentate ligands. The thermal behavior of some chelates is studied and the activation thermodynamic parameters are calculated using Coats-Redfern method. The corrosion inhibition of stainless steel types 410 and 304 in 1 M HCl using the synthesized Schiff bases as inhibitors have been studied by weight loss method. The obtained data considered these ligands as efficient corrosion inhibitors. The ligands and their metal complexes exhibited considerable antibacterial activity against Staphylococcusaureus, and Escherichiacoli and antifungal activity against Candida albicans.

  4. Cucurbitacin delta 23-reductase from the fruit of Cucurbita maxima var. Green Hubbard. Physicochemical and fluorescence properties and enzyme-ligand interactions.

    PubMed Central

    Dirr, H W; Schabort, J C; Weitz, C

    1986-01-01

    Cucurbitacin delta 23-reductase from Cucurbita maxima var. Green Hubbard fruit displays an apparent Mr of 32,000, a Stokes radius of 263 nm and a diffusion coefficient of 8.93 X 10(-7) cm2 X s-1. The enzyme appears to possess a homogeneous dimeric quaternary structure with a subunit Mr of 15,000. Two tryptophan and fourteen tyrosine residues per dimer were found. Emission spectral properties of the enzyme and fluorescence quenching by iodide indicate the tryptophan residues to be buried within the protein molecule. In the pH range 5-7, where no conformational changes were detected, protonation of a sterically related ionizable group with a pK of approx. 6.0 markedly influenced the fluorescence of the tryptophan residues. Protein fluorescence quenching was employed to determine the dissociation constants for binding of NADPH (Kd 17 microM), NADP+ (Kd 30 microM) and elaterinide (Kd 227 microM). Fluorescence energy transfer between the tryptophan residues and enzyme-bound NADPH was observed. Images Fig. 1. PMID:3707515

  5. High-yield functional expression of human sodium/d-glucose cotransporter1 in Pichia pastoris and characterization of ligand-induced conformational changes as studied by tryptophan fluorescence.

    PubMed

    Tyagi, Navneet K; Goyal, Pankaj; Kumar, Azad; Pandey, Dharmendra; Siess, Wolfgang; Kinne, Rolf K H

    2005-11-29

    Studies on the structure-function relationship of transporters require the availability of sufficient amounts of the protein in a functional state. In this paper, we report the functional expression, purification, and reconstitution of the human sodium/d-glucose cotransporter1 (hSGLT1) in Pichia pastoris and ligand-induced conformational changes of hSGLT1 in solution as studied by intrinsic tryptophan fluorescence. hSGLT1 gene containing FLAG tag at position 574 was cloned into pPICZB plasmid, and the resulting expression vector pPICZB-hSGLT1 was introduced into P. pastoris strain GS115 by electroporation. Purification of recombinant hSGLT1 by nickel-affinity chromatography yields about 3 mg of purified recombinant hSGLT1 per 1-liter of cultured Pichia cells. Purified hSGLT1 migrates on SDS-PAGE with an apparent mass of 55 kDa. Kinetic analysis of hSGLT1 in proteoliposomes revealed sodium-dependent, secondary active, phlorizin-sensitive, and stereospecific alpha-methyl-d-glucopyranoside transport, demonstrating its full catalytic activity. The position of the maximum intrinsic tryptophan fluorescence and titration with hydrophilic collisional quenchers KI, acrylamide, and trichloroethanol suggested that most of Trps in hSGLT1 in solution are in a hydrophobic environment. In the presence of sodium, sugars that have been identified earlier as substrate for the transporter increase intrinsic fluorescence in a saturable manner by a maximum of 15%. alpha-Methyl-d-glucopyranoside had the highest affinity (K(d) = 0.71 mM), followed by d-glucose, d-galactose, d-mannose, and d-allose which showed a much lower affinity. l-Glucose was without effect. d-Glucose also increased the accessibility of the Trps to hydrophilic collisional quenchers. On the contrary phlorizin, the well-established inhibitor of SGLT1, decreased intrinsic fluorescence by a maximum of 50%, and induced a blue shift of maximum (5 nm). Again, the effects were sodium-dependent and saturable and a high

  6. Modulation of Intracellular Quantum Dot to Fluorescent Protein Förster Resonance Energy Transfer via Customized Ligands and Spatial Control of Donor–Acceptor Assembly

    PubMed Central

    Field, Lauren D.; Walper, Scott A.; Susumu, Kimihiro; Oh, Eunkeu; Medintz, Igor L.; Delehanty, James B.

    2015-01-01

    Understanding how to controllably modulate the efficiency of energy transfer in Förster resonance energy transfer (FRET)-based assemblies is critical to their implementation as sensing modalities. This is particularly true for sensing assemblies that are to be used as the basis for real time intracellular sensing of intracellular processes and events. We use a quantum dot (QD) donor -mCherry acceptor platform that is engineered to self-assemble in situ wherein the protein acceptor is expressed via transient transfection and the QD donor is microinjected into the cell. QD-protein assembly is driven by metal-affinity interactions where a terminal polyhistidine tag on the protein binds to the QD surface. Using this system, we show the ability to modulate the efficiency of the donor–acceptor energy transfer process by controllably altering either the ligand coating on the QD surface or the precise location where the QD-protein assembly process occurs. Intracellularly, a short, zwitterionic ligand mediates more efficient FRET relative to longer ligand species that are based on the solubilizing polymer, poly(ethylene glycol). We further show that a greater FRET efficiency is achieved when the QD-protein assembly occurs free in the cytosol compared to when the mCherry acceptor is expressed tethered to the inner leaflet of the plasma membrane. In the latter case, the lower FRET efficiency is likely attributable to a lower expression level of the mCherry acceptor at the membrane combined with steric hindrance. Our work points to some of the design considerations that one must be mindful of when developing FRET-based sensing schemes for use in intracellular sensing. PMID:26690153

  7. Modulation of Intracellular Quantum Dot to Fluorescent Protein Förster Resonance Energy Transfer via Customized Ligands and Spatial Control of Donor-Acceptor Assembly.

    PubMed

    Field, Lauren D; Walper, Scott A; Susumu, Kimihiro; Oh, Eunkeu; Medintz, Igor L; Delehanty, James B

    2015-01-01

    Understanding how to controllably modulate the efficiency of energy transfer in Förster resonance energy transfer (FRET)-based assemblies is critical to their implementation as sensing modalities. This is particularly true for sensing assemblies that are to be used as the basis for real time intracellular sensing of intracellular processes and events. We use a quantum dot (QD) donor -mCherry acceptor platform that is engineered to self-assemble in situ wherein the protein acceptor is expressed via transient transfection and the QD donor is microinjected into the cell. QD-protein assembly is driven by metal-affinity interactions where a terminal polyhistidine tag on the protein binds to the QD surface. Using this system, we show the ability to modulate the efficiency of the donor-acceptor energy transfer process by controllably altering either the ligand coating on the QD surface or the precise location where the QD-protein assembly process occurs. Intracellularly, a short, zwitterionic ligand mediates more efficient FRET relative to longer ligand species that are based on the solubilizing polymer, poly(ethylene glycol). We further show that a greater FRET efficiency is achieved when the QD-protein assembly occurs free in the cytosol compared to when the mCherry acceptor is expressed tethered to the inner leaflet of the plasma membrane. In the latter case, the lower FRET efficiency is likely attributable to a lower expression level of the mCherry acceptor at the membrane combined with steric hindrance. Our work points to some of the design considerations that one must be mindful of when developing FRET-based sensing schemes for use in intracellular sensing. PMID:26690153

  8. Non-derivatization approach to high-performance liquid chromatography-fluorescence detection for aminoglycoside antibiotics based on a ligand displacement reaction.

    PubMed

    Yang, M; Tomellini, S A

    2001-12-21

    An indirect fluorescence detection method has been developed for detecting the aminoglycoside antibiotics following chromatographic separation. This approach to detection is based on a displacement reaction between the aminoglycosides and a copper(II)-L-tryptophan (L-Trp) complex, Cu(L-Trp)2. The aminoglycosides, which contain multiple amino groups, have strong affinities for the Cu(II) ion and displace L-Trp from the Cu(L-Trp)2 complex. The resulting increase in L-Trp fluorescence, which is quenched when coordinated to Cu(II), is indicative of the presence of the aminoglycoside. Fluorescence titration data indicate that there is a stoichiometric ratio of 1:1 between the reaction of the aminoglycosides with Cu(L-Trp)2. This HPLC detection scheme is implemented postcolumn by mixing a buffered Cu(L-Trp)2 solution with the column eluent prior to detection. The aminoglycosides were separated with the use of a column packed with a polymeric strong cation-exchanger. Separation and detection variables were optimized and are discussed. The detection limits for the aminoglycosides tested ranged from 4.2 to 14.5 ng injected (S/N=3). A linear working curve was achieved for amikacin in the range of 29-586 ng for a six point linearity test. The developed separation and detection scheme was further tested by analyzing commercial pharmaceutical formulations of these antibiotics. PMID:11806546

  9. Fluorescent Cross-Linked Supramolecular Polymer Constructed by Orthogonal Self-Assembly of Metal-Ligand Coordination and Host-Guest Interaction.

    PubMed

    Qian, Xiaomin; Gong, Weitao; Li, Xiaopeng; Fang, Le; Kuang, Xiaojun; Ning, Guiling

    2016-05-10

    A new host molecule consists of four terpyridine groups as the binding sites with zinc(II) ion and a copillar[5]arene incorporated in the center as a spacer to interact with guest molecule was designed and synthesized. Due to the 120 ° angle of the rigid aromatic segment, a cross-linked dimeric hexagonal supramolecular polymer was therefore generated as the result of the orthogonal self-assembly of metal-ligand coordination and host-guest interaction. UV/Vis spectroscopy, (1) H NMR spectroscopy, viscosity and dynamic light-scattering techniques were employed to characterize and understand the cross-linking process with the introduction of zinc(II) ion and guest molecule. More importantly, well-defined morphology of the self-assembled supramolecular structure can be tuned by altering the adding sequence of the two components, that is, the zinc(II) ion and the guest molecule. In addition, introduction of a competitive ligand suggested the dynamic nature of the supramolecular structure. PMID:27062539

  10. New tungstenocenes containing 3-hydroxy-4-pyrone ligands: antiproliferative activity on HT-29 and MCF-7 cell lines and binding to human serum albumin studied by fluorescence spectroscopy and molecular modeling methods

    PubMed Central

    Domínguez-García, Moralba; Ortega-Zúñiga, Carlos; Meléndez, Enrique

    2012-01-01

    Three new water-soluble tungstenocene derivatives were synthesized and characterized using 3-hydroxy-4-pyrone ligands, which provide aqueous stability to the complexes. The antiproliferative activities of the complexes on HT-29 colon cancer and MCF-7 breast cancer cell lines were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and showed the new tungstenocene derivatives have higher antiproliferative action than tungstenocene dichloride (Cp2WCl2, where Cp is cyclopentadienyl). The binding interactions of the tungstenocenes with human serum albumin (HSA) were investigated using fluorescence spectroscopy and molecular modeling methods. Analysis of the fluorescence quenching spectra indicates that the tungstenocene complexes bind HSA by hydrophobic interactions and hydrogen bonding at fatty acid binding site 6 and drug binding site II. Docking studies provided a description of the hydrophobic interactions and hydrogen bonding by which the tungstenocenes become engaged with HSA. It was determined that the binding affinity of the tungstenoecenes for HSA is in the order Cp2WCl2 < [Cp2W(ethyl maltolato)]Cl < [Cp2W (maltolato)]Cl < [Cp2W(kojato)]Cl, consistent with the hydrophobic interactions and the number of hydrogen bonds involved. PMID:23212785

  11. Binding of a fluorescence reporter and a ligand to an odorant-binding protein of the yellow fever mosquito, Aedes aegypti.

    PubMed

    Leal, Gabriel M; Leal, Walter S

    2014-01-01

    Odorant-binding proteins (OBPs), also named pheromone-binding proteins when the odorant is a pheromone, are essential for insect olfaction. They solubilize odorants that reach the port of entry of the olfactory system, the pore tubules in antennae and other olfactory appendages. Then, OBPs transport these hydrophobic compounds through an aqueous sensillar lymph to receptors embedded on dendritic membranes of olfactory receptor neurons. Structures of OBPs from mosquito species have shed new light on the mechanism of transport, although there is considerable debate on how they deliver odorant to receptors. An OBP from the southern house mosquito, Culex quinquefasciatus, binds the hydrophobic moiety of a mosquito oviposition pheromone (MOP) on the edge of its binding cavity. Likewise, it has been demonstrated that the orthologous protein from the malaria mosquito binds the insect repellent DEET on a similar edge of its binding pocket. A high school research project was aimed at testing whether the orthologous protein from the yellow fever mosquito, AaegOBP1, binds DEET and other insect repellents, and MOP was used as a positive control. Binding assays using the fluorescence reporter N-phenyl-1-naphtylamine (NPN) were inconclusive. However, titration of NPN fluorescence emission in AaegOBP1 solution with MOP led to unexpected and intriguing results. Quenching was observed in the initial phase of titration, but addition of higher doses of MOP led to a stepwise increase in fluorescence emission coupled with a blue shift, which can be explained at least in part by formation of MOP micelles to house stray NPN molecules. PMID:25671088

  12. Binding of a fluorescence reporter and a ligand to an odorant-binding protein of the yellow fever mosquito, Aedes aegypti

    PubMed Central

    Leal, Gabriel M.; Leal, Walter S.

    2015-01-01

    Odorant-binding proteins (OBPs), also named pheromone-binding proteins when the odorant is a pheromone, are essential for insect olfaction. They solubilize odorants that reach the port of entry of the olfactory system, the pore tubules in antennae and other olfactory appendages. Then, OBPs transport these hydrophobic compounds through an aqueous sensillar lymph to receptors embedded on dendritic membranes of olfactory receptor neurons. Structures of OBPs from mosquito species have shed new light on the mechanism of transport, although there is considerable debate on how they deliver odorant to receptors. An OBP from the southern house mosquito, Culex quinquefasciatus, binds the hydrophobic moiety of a mosquito oviposition pheromone (MOP) on the edge of its binding cavity. Likewise, it has been demonstrated that the orthologous protein from the malaria mosquito binds the insect repellent DEET on a similar edge of its binding pocket. A high school research project was aimed at testing whether the orthologous protein from the yellow fever mosquito, AaegOBP1, binds DEET and other insect repellents, and MOP was used as a positive control. Binding assays using the fluorescence reporter N-phenyl-1-naphtylamine (NPN) were inconclusive. However, titration of NPN fluorescence emission in AaegOBP1 solution with MOP led to unexpected and intriguing results. Quenching was observed in the initial phase of titration, but addition of higher doses of MOP led to a stepwise increase in fluorescence emission coupled with a blue shift, which can be explained at least in part by formation of MOP micelles to house stray NPN molecules. PMID:25671088

  13. Assembly multi-dimensional CdII coordination architectures based on flexible bis(benzimidazole) ligands: Diversity of their coordination geometries and fluorescent properties

    NASA Astrophysics Data System (ADS)

    Jiao, Cui-huan; Geng, Jian-chen; He, Cui-hong; Cui, Guang-hua

    2012-08-01

    Based on three structurally related flexible bis(5,6-dimethylbenzimidazole) ligand, five novel metal-organic CdII coordination architectures: from 0D to 3D structures CdII complexes have been hydrothermally synthesized and structurally characterized, namely, Cd2I4(L1)2 (1), [CdCl2(L1)]n (2), [CdCl2(L2)]n (3), {[Cd(chdc)(L2)0.5]·H2O}n (4), {[Cd(pydca)(L3)0.5(H2O)2]·H2O}n (5) (where L1 = 1,2-bis(5,6-dimethylbenzimidazole)ethane, L2 = 1,3-bis(5,6-dimethylbenzimidazole)propane, L3 = 1,4-bis(5,6-dimethylbenzimidazole)butane, H2chdc = 1,4-cyclohexanedicarboxylic acid, H2pydca = pyridine-2,6-dicarboxylic acid). A discrete binuclear [2 + 2] metallomacrocycles cadmium(II) complex of 1 is 0D, 3 and 5 exhibit one-dimensional helical and zigzag chain structures, respectively. 4 Forms a 2D layer with sql net topology bridged by carboxylate anion and L2, while 2 is an overall 3D array with the diamond topology (dia). In these complexes, the influences of anions coordination on the framework formation were observed and discussed. These results indicate the spacer length of the ligands and anions play important roles in controlling the diversity structural topologies of such metal-organic coordination architectures. The thermogravimetric analyses, X-ray powder diffraction and solid-state luminescent properties of the complexes have also been investigated.

  14. Biointeractions of C.I. Acid Red 2 and its structural analogues with transporter albumin: Fluorescence, circular dichroism, and ligand docking approaches.

    PubMed

    Peng, Wei; Ding, Fei; Xie, Yong

    2016-01-01

    In this contribution, the toxicological effects of C.I. Acid Red 2 and 1-(2-pyridylazo)-2-naphthol (PAN) have been elucidated by utilizing plasma albumin as a biological model. Fluorescence data indicated that the Trp-214 residue was quenched by both azo compounds, but the quenching degree of C.I. Acid Red 2 is less than PAN. According to the results of time-resolved fluorescence decay, it may be observed that the quenching of Trp-214 residue is controlled by static type; this corroborates the Stern-Volmer analyses and the conformational transition of protein was concurred. The experiments also found that azo colorants are situated within subdomain IIA, several amino acid residues, such as Ser-202, Ala-210, and Trp-214 were believed to be yielded direct interaction with the two chemicals, yet the operating distances between C.I. Acid Red 2 and relevant residues are greater than PAN. Interestingly, we may ascertain that the azo colorants with naphthalene ring possess stronger affinity with protein than those just having benzene ring in their molecular structure. This suggested that the existence of naphthalene ring substituent could hold relatively great risk for the human body due to large hydrophobicity (cLogP); therefore, the hydrophobicity of azo colorants can probably be a major element of its toxicological activities. PMID:26682933

  15. Detection of nearest neighbors to specific fluorescently tagged ligands in rod outer segment and lymphocyte plasma membranes by photosensitization of 5-iodonaphthyl 1-azide

    SciTech Connect

    Raviv, Y.; Bercovici, T.; Gitler, C.; Salomon, Y. )

    1989-02-07

    Lima bean agglutinin-fluorescein 5-isothiocyanate conjugate (FluNCS-lima bean lectin) interacts with specific receptor molecules on membranes both from the rod outer segment (ROS) of the frog retina and from S49 mouse lymphoma cells. When (125I)-5-iodonaphthyl 1-azide (125I-INA), which freely and randomly partitions into the lipid bilayer, is added to membranes and the suspension is irradiated at 480 nm, the FluNCS-conjugated lectin photosensitizes the (125I)INA but only at discrete sites. This results in the selective labeling of specific proteins: an 88-kDa protein on ROS membranes and a 56-kDa protein on S49 plasma membranes. Labeling is dependent upon the interaction of the FluNCS-lectin with glycosylated receptor sites, since N-acetylgalactosamine, but not methyl alpha-mannoside, blocked labeling of the 56-kDa protein on S49 membranes. In contrast, a random labeling pattern of membrane proteins was observed upon irradiation at 480 nm using other fluorescein conjugates, such as FluNCS-bovine serum albumin (FluNCS-BSA) or FluNCS-soybean trypsin inhibitor (FluNCS-STI), which interact with cell membranes in a nonselective manner, or with N-(fluorescein-5-thiocarbamoyl)-n-undecyclamine (FluNCS-NHC11), which is freely miscible in the membrane lipid. Random labeling was also obtained by direct photoexcitation of (125I)INA at 314 nm, with no distinct labeling of the 88- and 56-kDa proteins in the respective membranes. These results suggest that protein ligands can be used to guide sensitizers to discrete receptor sites and lead to their selective labeling by photosensitized activation of (125I)INA.

  16. Improved synthesis of DOTA tetraamide lanthanide(III) ligands: tool for increasing the repertoire of potential PARACEST contrast agents for MRI and/or fluorescent sensors

    PubMed Central

    De León-Rodríguez, Luis M.; Viswanathan, Subha; Sherry, A. Dean

    2011-01-01

    The synthesis of new DOTA tetra-amide (DOTAMR4) compounds is of great interest given their application in the formation of Ln(III) complexes which are potential PARACEST contrast agents in MRI or fluorescent molecular probes. In this context amino acid and peptide DOTAMR4 derivatives are particularly attractive since the amino-acid and/or peptide moiety can show responsive properties dependent on a given stimuli which might translate to changes in water exchange rates of the corresponding Ln(III) complex. Current synthesis of DOTAMR4 derivatives is typically carried out by reacting haloacetamide intermediates with cyclen. However, this method fails to generate the tetra-substituted products when bulky substituents are present in the haloacetamide and in some cases this intermediate cannot be prepared by conventional acylation procedures limiting the number of DOTAMR4 compounds available for study. As a solution to these limitations, an improved methodology for the synthesis of DOTAMR4 by coupling DOTA to an appropriate amine containing reagent (i.e. protected amino-acids with the α-amino group free) is presented in this work. Several DOTAMR4 derivatives which are difficult or impossible to prepare with the traditional methodologies were easily obtained starting with DOTA. A new protocol was derived using this methodology for the solution phase synthesis of DOTA peptide derivatives. With this methodology, many other DOTAMR4 peptide and non-peptide derivatives have been prepared in our laboratories with several of these new compounds showing interesting properties for molecular imaging. PMID:20586036

  17. Glyconanomaterials: Synthesis, Characterization, and Ligand Presentation

    PubMed Central

    Wang, Xin

    2010-01-01

    Glyconanomaterials, nanomaterials carrying surface-tethered carbohydrate ligands, have emerged and demonstrated increasing potential in biomedical imaging, therapeutics, and diagnostics. These materials combine the unique properties of nanometer-scale objects with the ability to present multiple copies of carbohydrate ligands, greatly enhancing the weak affinity of individual ligands to their binding partners. Critical to the performance of glyconanomaterials is the proper display of carbohydrate ligands, taking into consideration of the coupling chemistry, the type and length of the spacer linkage, and the ligand density. This article provides an overview of the coupling chemistry for attaching carbohydrate ligands to nanomaterials, and discusses the need for thorough characterization of glyconanomaterials, especially quantitative analyses of the ligand density and binding affinities. Using glyconanoparticles synthesized by a versatile photocoupling chemistry, methods for determining the ligand density by colorimetry and the binding affinity with lectins by a fluorescence competition assay are determined. The results show that the multivalent presentation of carbohydrate ligands significantly enhances the binding affinity by several orders of magnitude in comparison to the free ligands in solution. The effect is sizeable even at low surface ligand density. The type and length of the spacer linkage also affect the binding affinity, with the longer linkage promoting the association of bound ligands with the corresponding lectins. PMID:20301131

  18. Further Insights into Metal-DOM Interaction: Consideration of Both Fluorescent and Non-Fluorescent Substances

    PubMed Central

    Xu, Huacheng; Zhong, Jicheng; Yu, Guanghui; Wu, Jun; Jiang, Helong; Yang, Liuyan

    2014-01-01

    Information on metal binding with fluorescent substances has been widely studied. By contrast, information on metal binding with non-fluorescent substances remains lacking despite the dominance of these substances in aquatic systems. In this study, the metal binding properties of both fluorescent and non-fluorescent substances were investigated by using metal titration combined with two-dimensional correlation spectroscopy (2D–COS) analysis. The organic matters in the eutrophic algae-rich lake, including natural organic matters (NOM) and algae-induced extracellular polymeric substances (EPS), both contained fluorescent and non-fluorescent substances. The peaks in the one-dimensional spectra strongly overlapped, while 2D–COS can decompose the overlapped peaks and thus enhanced the spectral resolution. Moreover, 2D FTIR COS demonstrated that the binding susceptibility of organic ligands in both NOM and algal EPS matrices followed the order: 3400>1380>1650 cm−1, indicative the significant contribution of non-fluorescent ligands in metal binding. The modified Stern-Volmer equation also revealed a substantial metal binding potential for the non-fluorescent substances (logKM: 3.57∼4.92). As for the effects of organic ligands on metal binding, EPS was characterized with higher binding ability than NOM for both fluorescent and non-fluorescent ligands. Algae-induced EPS and the non-fluorescent substances in eutrophic algae-rich lakes should not be overlooked because of their high metal binding potential. PMID:25380246

  19. Fluorescence Analysis of Sulfonamide Binding to Carbonic Anhydrase

    ERIC Educational Resources Information Center

    Wang, Sheila C.; Zamble, Deborah B.

    2006-01-01

    A practical laboratory experiment is described that illustrates the application of fluorescence resonance energy transfer to the study of protein-ligand binding. The affinities of wild-type and mutant human carbonic anhydrase II for dansylamide were determined by monitoring the increase in ligand fluorescence that occurs due to energy transfer…

  20. Mapping Protein–Protein Interactions of the Resistance-Related Bacterial Zeta Toxin–Epsilon Antitoxin Complex (ε2ζ2) with High Affinity Peptide Ligands Using Fluorescence Polarization

    PubMed Central

    Fernández-Bachiller, María Isabel; Brzozowska, Iwona; Odolczyk, Norbert; Zielenkiewicz, Urszula; Zielenkiewicz, Piotr; Rademann, Jörg

    2016-01-01

    Toxin–antitoxin systems constitute a native survival strategy of pathogenic bacteria and thus are potential targets of antibiotic drugs. Here, we target the Zeta–Epsilon toxin–antitoxin system, which is responsible for the stable maintenance of certain multiresistance plasmids in Gram-positive bacteria. Peptide ligands were designed on the basis of the ε2ζ2 complex. Three α helices of Zeta forming the protein–protein interaction (PPI) site were selected and peptides were designed conserving the residues interacting with Epsilon antitoxin while substituting residues binding intramolecularly to other parts of Zeta. Designed peptides were synthesized with an N-terminal fluoresceinyl-carboxy-residue for binding assays and provided active ligands, which were used to define the hot spots of the ε2ζ2 complex. Further shortening and modification of the binding peptides provided ligands with affinities <100 nM, allowing us to determine the most relevant PPIs and implement a robust competition binding assay. PMID:27438853

  1. Mapping Protein-Protein Interactions of the Resistance-Related Bacterial Zeta Toxin-Epsilon Antitoxin Complex (ε₂ζ₂) with High Affinity Peptide Ligands Using Fluorescence Polarization.

    PubMed

    Fernández-Bachiller, María Isabel; Brzozowska, Iwona; Odolczyk, Norbert; Zielenkiewicz, Urszula; Zielenkiewicz, Piotr; Rademann, Jörg

    2016-01-01

    Toxin-antitoxin systems constitute a native survival strategy of pathogenic bacteria and thus are potential targets of antibiotic drugs. Here, we target the Zeta-Epsilon toxin-antitoxin system, which is responsible for the stable maintenance of certain multiresistance plasmids in Gram-positive bacteria. Peptide ligands were designed on the basis of the ε₂ζ₂ complex. Three α helices of Zeta forming the protein-protein interaction (PPI) site were selected and peptides were designed conserving the residues interacting with Epsilon antitoxin while substituting residues binding intramolecularly to other parts of Zeta. Designed peptides were synthesized with an N-terminal fluoresceinyl-carboxy-residue for binding assays and provided active ligands, which were used to define the hot spots of the ε₂ζ₂ complex. Further shortening and modification of the binding peptides provided ligands with affinities <100 nM, allowing us to determine the most relevant PPIs and implement a robust competition binding assay. PMID:27438853

  2. Fluorescent Aptamer Sensors

    NASA Astrophysics Data System (ADS)

    Chen, Hui William; Kim, Youngmi; Meng, Ling; Mallikaratchy, Prabodhika; Martin, Jennifer; Tang, Zhiwen; Shangguan, Dihua; O'Donoghue, Meghan; Tan, Weihong

    Aptamers are single-stranded nucleic acid probes that can be evolved to have high specificity and affinity for different targets. These targets include biomar-ker proteins, small molecules, and even whole live cells that express a variety of surface proteins of interest. Aptamers offer several advantages over protein-based molecular probes such as low immunogenic activity, flexible modification, and in vitro synthesis. In addition, aptamers used as molecular probes can be made with easy signaling for binding with their corresponding targets. There are a few different fluorescence-based signal transduction mechanisms, such as direct fluorophore labeling, fluorescence resonance energy transfer (FRET), fluorescence quenching, fluorescence anisotropy, and light-switching excimers. These signaling processes in combination with various labeling strategies of nucleic acid aptamers contribute to simple, rapid, sensitive, and selective biological assays. In this chapter, we discuss the optical signaling of aptamers for single proteins such as α-thrombin and platelet-derived growth factor (PDGF). We also present detailed discussion about fluorescent aptamers developed from cell-based systematic evolution of ligands by exponential enrichment (SELEX) for the recognition of different target tumor cells.

  3. Synthesis, structures and fluorescent properties of two novel lanthanide [Ln = Ce(III), Pr(III)] coordination polymers based on 1,3-benzenedicarboxylate and 2-(4-methoxyphenyl)-1H-imidazo[4,5-f][1,10]phenanthroline ligands

    NASA Astrophysics Data System (ADS)

    Wang, Lei; Ni, Liang; yao, Jia

    2012-09-01

    Two structurally diverse coordination polymers [Ce2(m-BDC)2(m-HBDC)2(MOPIP)2·3/2H2O]n (1) and [Pr2(m-BDC)3(MOPIP)2·H2O]n(2) have been synthesized by hydrothermal reaction of lanthanide chloride with mixed ligands benzene-1,3-dicarboxylic acid and 2-(4-methoxyphenyl)-1H-imidazo[4,5-f][1,10]phenanthroline (MOPIP). The crystal structures of the complexes are zipper-like chains of octacoordinate Ln3+ ions, in which Ln3+ ions are bridged in different coordination modes by m-BDC2+ and decorated by MOPIP ligands. These chains are further assembled into three-dimensional supramolecular framework by π⋯π stacking and hydrogen bonding interactions. The fluorescent property and thermal stability were also investigated. Additionally, Natural bond orbital (NBO) analysis of complex 2 shows a weak covalent interaction between the coordinated atoms and Pr3+ ions.

  4. Metal-ligand cooperation.

    PubMed

    Khusnutdinova, Julia R; Milstein, David

    2015-10-12

    Metal-ligand cooperation (MLC) has become an important concept in catalysis by transition metal complexes both in synthetic and biological systems. MLC implies that both the metal and the ligand are directly involved in bond activation processes, by contrast to "classical" transition metal catalysis where the ligand (e.g. phosphine) acts as a spectator, while all key transformations occur at the metal center. In this Review, we will discuss examples of MLC in which 1) both the metal and the ligand are chemically modified during bond activation and 2) bond activation results in immediate changes in the 1st coordination sphere involving the cooperating ligand, even if the reactive center at the ligand is not directly bound to the metal (e.g. via tautomerization). The role of MLC in enabling effective catalysis as well as in catalyst deactivation reactions will be discussed. PMID:26436516

  5. Application of BRET to monitor ligand binding to GPCRs

    PubMed Central

    Stoddart, Leigh A; Johnstone, Elizabeth K M; Wheal, Amanda J; Goulding, Joëlle; Robers, Matthew B; Machleidt, Thomas; Wood, Keith V

    2015-01-01

    Bioluminescence resonance energy transfer (BRET) is a well-established method for investigating protein-protein interactions. Here we present a novel BRET approach to monitor ligand binding to G protein-coupled receptors (GPCRs) on the surface of living cells made possible by the use of fluorescent ligands in combination with a novel bioluminescent protein (NanoLuc) that can be readily expressed on the N-terminus of GPCRs. PMID:26030448

  6. Application of BRET to monitor ligand binding to GPCRs.

    PubMed

    Stoddart, Leigh A; Johnstone, Elizabeth K M; Wheal, Amanda J; Goulding, Joëlle; Robers, Matthew B; Machleidt, Thomas; Wood, Keith V; Hill, Stephen J; Pfleger, Kevin D G

    2015-07-01

    Bioluminescence resonance energy transfer (BRET) is a well-established method for investigating protein-protein interactions. Here we present a BRET approach to monitor ligand binding to G protein-coupled receptors (GPCRs) on the surface of living cells made possible by the use of fluorescent ligands in combination with a bioluminescent protein (NanoLuc) that can be readily expressed on the N terminus of GPCRs. PMID:26030448

  7. A screening cascade to identify ERβ ligands

    PubMed Central

    Filgueira, Carly S.; Benod, Cindy; Lou, Xiaohua; Gunamalai, Prem S.; Villagomez, Rosa A.; Strom, Anders; Gustafsson, Jan-Åke; Berkenstam, Anders L.; Webb, Paul

    2014-01-01

    The establishment of effective high throughput screening cascades to identify nuclear receptor (NR) ligands that will trigger defined, therapeutically useful sets of NR activities is of considerable importance. Repositioning of existing approved drugs with known side effect profiles can provide advantages because de novo drug design suffers from high developmental failure rates and undesirable side effects which have dramatically increased costs. Ligands that target estrogen receptor β (ERβ) could be useful in a variety of diseases ranging from cancer to neurological to cardiovascular disorders. In this context, it is important to minimize cross-reactivity with ERα, which has been shown to trigger increased rates of several types of cancer. Because of high sequence similarities between the ligand binding domains of ERα and ERβ, preferentially targeting one subtype can prove challenging. Here, we describe a sequential ligand screening approach comprised of complementary in-house assays to identify small molecules that are selective for ERβ. Methods include differential scanning fluorimetry, fluorescence polarization and a GAL4 transactivation assay. We used this strategy to screen several commercially-available chemical libraries, identifying thirty ERβ binders that were examined for their selectivity for ERβ versus ERα, and tested the effects of selected ligands in a prostate cancer cell proliferation assay. We suggest that this approach could be used to rapidly identify candidates for drug repurposing. PMID:25422593

  8. Chelating ligands for nanocrystals' surface functionalization.

    PubMed

    Querner, Claudia; Reiss, Peter; Bleuse, Joël; Pron, Adam

    2004-09-22

    A new family of ligands for the surface functionalization of CdSe nanocrystals is proposed, namely alkyl or aryl derivatives of carbodithioic acids (R-C(S)SH). The main advantages of these new ligands are as follows: they nearly quantitatively exchange the initial surface ligands (TOPO) in very mild conditions; they significantly improve the resistance of nanocrystals against photooxidation because of their ability of strong chelate-type binding to metal atoms; their relatively simple preparation via Grignard intermediates facilitates the development of new bifunctional ligands containing, in addition to the anchoring carbodithioate group, a second function, which enables the grafting of molecules or macromolecules of interest on the nanocrystal surface. To give an example of this approach, we report, for the first time, the grafting of an electroactive oligomer from the polyaniline family-aniline tetramer-on CdSe nanocrystals after their functionalization with 4-formyldithiobenzoic acid. The grafting proceeds via a condensation reaction between the aldehyde group of the ligand and the terminal primary amine group of the tetramer. The resulting organic/inorganic hybrid exhibits complete extinction of the fluorescence of its constituents, indicating efficient charge or energy transfer between the organic and the inorganic semiconductors. PMID:15366904

  9. LIGAND-INDUCED CHANGES IN T BOX ANTITERMINATOR RNA STABILITY

    PubMed Central

    Zhou, S.; Acquaah-Harrison, G.; Jack, K.D.; Bergmeier, S.C.; Hines, J.V.

    2012-01-01

    The T box antiterminator RNA element is an important component of the T box riboswitch that controls the transcription of vital genes in many Gram-positive bacteria. A series of 1,4-disubstituted 1,2,3-triazoles was screened in a fluorescence-monitored thermal denaturation assay to identify ligands that altered the stability of antiterminator model RNA. Several ligands were identified that significantly increased or decreased the melting temperature (Tm) of the RNA. The results indicate that this series of triazole ligands can alter the stability of antiterminator model RNA in a structure-dependent manner. PMID:22117759

  10. Polyethylene glycol-based homologated ligands for nicotinic acetylcholine receptors☆

    PubMed Central

    Scates, Bradley A.; Lashbrook, Bethany L.; Chastain, Benjamin C.; Tominaga, Kaoru; Elliott, Brandon T.; Theising, Nicholas J.; Baker, Thomas A.; Fitch, Richard W.

    2010-01-01

    A homologous series of polyethylene glycol (PEG) monomethyl ethers were conjugated with three ligand series for nicotinic acetylcholine receptors. Conjugates of acetylaminocholine, the cyclic analog 1-acetyl-4,4-dimethylpiperazinium, and pyridyl ether A-84543 were prepared. Each series was found to retain significant affinity at nicotinic receptors in rat cerebral cortex with tethers of up to six PEG units. Such compounds are hydrophilic ligands which may serve as models for fluorescent/affinity probes and multivalent ligands for nAChR. PMID:19006672

  11. Effects of ligand binding on the conformation and internal dynamics in specific regions of porcine pancreatic phospholipase A2 with tryptophan as a probe: a study combining time-resolved fluorescence spectroscopy and site-directed mutagenesis (same as p. 100)

    NASA Astrophysics Data System (ADS)

    Kuipers, Oscar; Vincent, Michel; Brochon, Jean-Claude; Verheij, Bert; de Haas, Gerard; Gallay, Jacques

    1990-05-01

    Exploration of the effect of ligand-protein interactions on conformational substates and internal dynamics in different regions of phospholipase A2 from porcine pancreas (PLA2), was performed by combining site-directed mutagenesis and time-resolved fluorescence measurements. The single tryptophan residue (Trp-3) in the wild type protein was replaced by a phenylalanine residue, whereafter Trp was substituted either for leucine-31 ,located in the calcium binding loop, or for phenylalanine-94, located at the "back side" of the enzyme, in a-helix E (Dijkstra et al., J. Mol. Biol., 147, 97-123, 1981). Analyses by the Maximum Entropy Method (MIEM) of the total fluorescence intensity decays, provide in each case a distribution of separate lifetime classes, which can be interpreted as reflecting the existence of discrete conformational substates in slow exchange with respect to the time-scale of the decay kinetics. The fluorescence decay of the W94 mutant is dominated by an extremely short excited state lifetime of ~60 ps, probably arising from the presence of two proximate disulfide bridges. Time-resolved fluorescence anisotropy studies show that the Trp residue near the NH2 terminus (Trp-3) undergoes a more limited rotational motion than the Trp-3 1 located in the calcium binding loop. The widest angular rotation is observed at position 94, in a-helix E. Calcium binding displays the strongest influence on the lifetime distribution of Trp-31: a major local conformation corresponding to a lifetime class with a barycenter value of ~5.5 ns and contributing to ~50% of the decay is selected. The conformations giving rise to the short lifetimes ((tau)1 and (tau)2 lifetime classes) become less important. The contribution of the third lifetime class (c3) stays at a constant value of 30%. In the presence of calcium, the amplitude of motion is wider than without the ion. There is virtually no effect of calcium binding on the lifetime distribution of the Trp residue at the 3 or the

  12. Effects of ligand binding on the conformation and internal dynamics in specific regions of porcine pancreatic phospholipase A2 with tryptophan as a probe: a study combinging time-resolved fluorescence spectroscopy and site-directed mutagenesis (same as p. 628)

    NASA Astrophysics Data System (ADS)

    Kuipers, Oscar; Vincent, Michel; Brochon, Jean-Claude; Verheij, Bert; de Haas, Gerard; Gallay, Jacques

    1990-05-01

    Exploration of the effect of ligand-protein interactions on conformational substates and internal dynamics in different regions of phospholipase A2 from porcine pancreas (PLA2), was performed by combining site-directed mutagenesis and time-resolved fluorescence measurements. The single tryptophan residue (Trp-3) in the wild type protein was replaced by a phenylalanine residue, whereafter Tip was substituted either for leucine-3 1 ,located in the calcium binding ioop, or for phenylalanine-94, located at the "back side" of the enzyme, in a-helix E (Dijkstra et al., J. Mol. Biol., 147, 97-123, 1981). Analyses by the Maximum Entropy Method (MEM) of the total fluorescence intensity decays, provide in each case a distribution of separate lifetime classes, which can be interpreted as reflecting the existence of discrete conformational substates in slow exchange with respect to the time-scale of the decay kinetics. The fluorescence decay of the W94 mutant is. dominated by an extremely short excited state lifetime of ~60 ps, probably arising from the presence of two proximate disulfide bridges. Time-resolved fluorescence anisotropy studies show that the Trp residue near the NH2 terminus (Trp-3) undergoes a more limited rotational motion than the Trp-3 1 located in the calcium binding loop. The widest angular rotation is observed at position 94, in a-helix E. Calcium binding displays the strongest influence on the lifetime distribution of Trp-3 1: a major local conformation corresponding to a lifetime class with a barycenter value of -5.5 ns and contributing to ~50% of the decay is selected. The conformations giving rise to the short lifetimes (τ1 and τ2 lifetime classes) become less important. The contribution of the third lifetime class (c3) stays at a constant value of 30%. In the presence of calcium, the amplitude of motion is wider than without the ion. There is virtually no effect of calcium binding on the lifetime distribution of the Trp residue at the 3 or the 94

  13. Saccharide sensing molecules having enhanced fluorescent properties

    DOEpatents

    Satcher Jr., Joe H.; Lane, Stephen M.; Darrow, Christopher B.; Cary, Douglas R.; Tran, Joe Anh

    2004-01-06

    The present invention provides formulae for fluorescent compounds that have a number of properties which make them uniquely suited for use in sensors of analytes such as saccharides. The advantageous fluorescent properties include favorable excitation wavelengths, emission wavelengths, fluorescence lifetimes, and photostability. Additional advantageous properties include enhanced aqueous solubility, as well as temperature and pH sensitivity. The compound comprises an aryl or a substituted phenyl botonic acid that acts as a substrate recognition component, a fluorescence switch component, and a fluorophore. Fluorescent compounds are described that are excited at wavelengths greater than 400 nm and emit at wavelengths greater than 450 nm, which is advantageous for optical transmission through skin. The fluorophore is typically selected from transition metal-ligand complexes and thiazine, oxazine, oxazone, or oxazine-one as well as anthracene compounds. The fluorescent compound can be immobilized in a glucose permeable biocompatible polymer matrix that is implantable below the skin.

  14. Synthesis of a new Cd(II)-Ni(II) hetero-metallic coordination polymer base on citric acid ligand. X-ray structure, thermal stability, XPS and fluorescence studies

    NASA Astrophysics Data System (ADS)

    Mtioui-Sghaier, O.; Mendoza-Meroño, R.; Fernández-Zapico, E.; García-Granda, S.; Fernández-González, A.; Ktari, L.; Dammak, M.

    2016-02-01

    A new hetero-metallic polymer, NiCd(cit)(H2O) (cit = citrate), was synthesized under hydrothermal conditions. The structural analysis indicates the formation of a 2D structure, bridged by the cit4- group. This compound crystallizes in the monoclinic P21/c space group, with lattice parameters: a = 6.0817(3) Å, b = 14.9725(6) Å, c = 9.6817(5) Å, β = 101.353(5)°. Full characterization by powder diffraction analysis, thermocalorimetry and scanning electron microscopy, XPS and fluorescence have been carried out. The TG-MS and DSC results show that this compound is thermally stable up to 250 °C and the DSC profile shows a medium-intense endotherm centred at approximately 300 °C. The distances of Cd-O are in the range of 2.169(4)-2.647(4) Å, similar to pure cadmium(II)-citrate aqueous complex Cd(C6H6O7)(H2O). The six-coordinated Cd2+ is linked by carboxylate groups to form an infinite chain, which is further connected through NiO6 octahedral bridges to generate 2D structure in the bc plane.

  15. On the origin of fluorescence in bacteriophytochrome infrared fluorescent proteins

    PubMed Central

    Samma, Alex A.; Johnson, Chelsea K.; Song, Shuang; Alvarez, Samuel

    2010-01-01

    Tsien (Science, 2009, 324, 804-807) has recently reported the creation of the first infrared fluorescent protein (IFP). It was engineered from bacterial phytochrome by removing the PHY and histidine kinase-related domains, by optimizing the protein to prevent dimerization and by limiting the biliverdins conformational freedom, especially around its D ring. We have used database analyses and molecular dynamics simulations with freely rotating chromophoric dihedrals in order to model the dihedral freedom available to the biliverdin D ring in the excited state; to show that the tetrapyrrole ligands in phytochromes are flexible and can adopt many conformations, however their conformational space is limited/defined by the chemospatial characteristics of the protein cavity. Our simulations confirm that the reduced accessibility to conformations geared to an excited state proton transfer may be responsible for the fluorescence in IFP, just as has been suggested by Kennis (PNAS, 2010, 107, 9170-9175) for fluorescent bacteriophytochrome from Rhodopseudomonas palustris. PMID:21047084

  16. Ligand modeling and design

    SciTech Connect

    Hay, B.P.

    1997-10-01

    The purpose of this work is to develop and implement a molecular design basis for selecting organic ligands that would be used in the cost-effective removal of specific radionuclides from nuclear waste streams. Organic ligands with metal ion specificity are critical components in the development of solvent extraction and ion exchange processes that are highly selective for targeted radionuclides. The traditional approach to the development of such ligands involves lengthy programs of organic synthesis and testing, which in the absence of reliable methods for screening compounds before synthesis, results in wasted research effort. The author`s approach breaks down and simplifies this costly process with the aid of computer-based molecular modeling techniques. Commercial software for organic molecular modeling is being configured to examine the interactions between organic ligands and metal ions, yielding an inexpensive, commercially or readily available computational tool that can be used to predict the structures and energies of ligand-metal complexes. Users will be able to correlate the large body of existing experimental data on structure, solution binding affinity, and metal ion selectivity to develop structural design criteria. These criteria will provide a basis for selecting ligands that can be implemented in separations technologies through collaboration with other DOE national laboratories and private industry. The initial focus will be to select ether-based ligands that can be applied to the recovery and concentration of the alkali and alkaline earth metal ions including cesium, strontium, and radium.

  17. Fluorescent refrigeration

    DOEpatents

    Epstein, Richard I.; Edwards, Bradley C.; Buchwald, Melvin I.; Gosnell, Timothy R.

    1995-01-01

    Fluorescent refrigeration is based on selective radiative pumping, using substantially monochromatic radiation, of quantum excitations which are then endothermically redistributed to higher energies. Ultimately, the populated energy levels radiatively deexcite emitting, on the average, more radiant energy than was initially absorbed. The material utilized to accomplish the cooling must have dimensions such that the exciting radiation is strongly absorbed, but the fluorescence may exit the material through a significantly smaller optical pathlength. Optical fibers and mirrored glasses and crystals provide this requirement.

  18. Photophysical study of the Ca 2+-chelator QUIN 2 ligand: effect of divalent and trivalent cations

    NASA Astrophysics Data System (ADS)

    Guardigli, M.; Sabbatini, N.

    1991-05-01

    The photophysical properties of complexes of the Ca 2+-chelator QUIN 2 ligand with divalent and trivalent cations have been studied. The absorption of the ligand is almost independent of the nature of the complexing cations, while the fluorescence emission strongly depends on the electric charge of the cations. Metal emission upon excitation in the ligand has been observed for the Eu 3+ complex, but not for the Tb 3+ complex.

  19. Ligand modeling and design

    SciTech Connect

    Hay, B.

    1996-10-01

    The purpose of this work is to develop and implement a molecular design basis for selecting organic ligands that would be used tin applications for the cost-effective removal of specific radionuclides from nuclear waste streams.

  20. Small Molecule Ligands for Bulged RNA Secondary Structures

    PubMed Central

    Meyer, S. Todd; Hergenrother, Paul J.

    2016-01-01

    A class of wedge-shaped small molecules has been designed, synthesized, and shown to bind bulged RNA secondary structures. These minimally cationic ligands exhibit good affinity and selectivity for certain RNA bulges as demonstrated in a fluorescent intercalator displacement assay. PMID:19678613

  1. Intracellular distribution of fluorescent copper and zinc bis(thiosemicarbazonato) complexes measured with fluorescence lifetime spectroscopy.

    PubMed

    Hickey, James L; James, Janine L; Henderson, Clare A; Price, Katherine A; Mot, Alexandra I; Buncic, Gojko; Crouch, Peter J; White, Jonathan M; White, Anthony R; Smith, Trevor A; Donnelly, Paul S

    2015-10-01

    The intracellular distribution of fluorescently labeled copper and zinc bis(thiosemicarbazonato) complexes was investigated in M17 neuroblastoma cells and primary cortical neurons with a view to providing insights into the neuroprotective activity of a copper bis(thiosemicarbazonato) complex known as Cu(II)(atsm). Time-resolved fluorescence measurements allowed the identification of the Cu(II) and Zn(II) complexes as well as the free ligand inside the cells by virtue of the distinct fluorescence lifetime of each species. Confocal fluorescent microscopy of cells treated with the fluorescent copper(II)bis(thiosemicarbazonato) complex revealed significant fluorescence associated with cytoplasmic puncta that were identified to be lysosomes in primary cortical neurons and both lipid droplets and lysosomes in M17 neuroblastoma cells. Fluorescence lifetime imaging microscopy confirmed that the fluorescence signal emanating from the lipid droplets could be attributed to the copper(II) complex but also that some degree of loss of the metal ion led to diffuse cytosolic fluorescence that could be attributed to the metal-free ligand. The accumulation of the copper(II) complex in lipid droplets could be relevant to the neuroprotective activity of Cu(II)(atsm) in models of amyotrophic lateral sclerosis and Parkinson's disease. PMID:26397162

  2. Extracellular interactions and ligand degradation shape the nodal morphogen gradient

    PubMed Central

    Wang, Yin; Wang, Xi; Wohland, Thorsten; Sampath, Karuna

    2016-01-01

    The correct distribution and activity of secreted signaling proteins called morphogens is required for many developmental processes. Nodal morphogens play critical roles in embryonic axis formation in many organisms. Models proposed to generate the Nodal gradient include diffusivity, ligand processing, and a temporal activation window. But how the Nodal morphogen gradient forms in vivo remains unclear. Here, we have measured in vivo for the first time, the binding affinity of Nodal ligands to their major cell surface receptor, Acvr2b, and to the Nodal inhibitor, Lefty, by fluorescence cross-correlation spectroscopy. We examined the diffusion coefficient of Nodal ligands and Lefty inhibitors in live zebrafish embryos by fluorescence correlation spectroscopy. We also investigated the contribution of ligand degradation to the Nodal gradient. We show that ligand clearance via degradation shapes the Nodal gradient and correlates with its signaling range. By computational simulations of gradient formation, we demonstrate that diffusivity, extra-cellular interactions, and selective ligand destruction collectively shape the Nodal morphogen gradient. DOI: http://dx.doi.org/10.7554/eLife.13879.001 PMID:27101364

  3. Fluorescent Human EP3 Receptor Antagonists.

    PubMed

    Tomasch, Miriam; Schwed, J Stephan; Kuczka, Karina; Meyer Dos Santos, Sascha; Harder, Sebastian; Nüsing, Rolf M; Paulke, Alexander; Stark, Holger

    2012-09-13

    Exchange of the lipophilc part of ortho-substituted cinnamic acid lead structures with different small molecule fluorophoric moieties via a dimethylene spacer resulted in hEP3R ligands with affinities in the nanomolar concentration range. Synthesized compounds emit fluorescence in the blue, green, and red range of light and have been tested concerning their potential as a pharmacological tool. hEP3Rs were visualized by confocal laser scanning microscopy on HT-29 cells, on murine kidney tissues, and on human brain tissues and functionally were characterized as antagonists on human platelets. Inhibition of PGE2 and collagen-induced platelet aggregation was measured after preincubation with novel hEP3R ligands. The pyryllium-labeled ligand 8 has been shown as one of the most promising structures, displaying a useful fluorescence and highly affine hEP3R antagonists. PMID:24900547

  4. Fluorescent noble metal nanoclusters

    NASA Astrophysics Data System (ADS)

    Zheng, Jie

    Water-soluble fluorescent metallic clusters at sizes comparable to the Fermi wavelength of an electron (˜0.5 nm for gold and silver) were created and their photophysical properties were investigated at the bulk and single molecule levels. We employed biocompatible dendrimer and peptide to prepare a series of strong fluorescent gold and silver clusters with chemical or photo reduction methods. Facilitated by the well-defined dendrimer size, electrospray ionization mass spectrometry indicates that the fluorescent silver nanocluster size ranges from 2 to 8 Ag atoms. The correlation of emission energy with the number of atoms, N, in each gold nanocluster is quantitatively fit for the smallest nanoclusters with no adjustable parameters by the simple scaling relation of EFermi/N1/3, in which EFermi is the Fermi energy of bulk gold. The transition energy scaling inversely with cluster radius indicates that electronic structure can be well described with the spherical jellium model and further demonstrates that these nanomaterials are "multi-electron artificial atoms". Fluorescence from these small metal clusters can be considered protoplasmonic, molecular transitions of the free conduction electrons before the onset of collective dipole oscillations occurring when a continuous density of states is reached. In addition, very strong single molecular Stokes and anti-Stokes Raman enhancement by fluorescent silver clusters was observed. Pushing to larger sizes, we also created ˜2nm diameter glutathione encapsulated luminescent gold nanoparticles. Distinct from similarly sized but nonluminescent gold nanoparticles, these 2 nm gold nanoparticles show bright, long lifetime emission but no plasmon absorption. The emission might arise from charge transfer between gold atoms and the thiol ligand. Providing the "missing link" between atomic and nanoparticle behavior in noble metals, these highly fluorescent, water-soluble gold and silver nanoclusters offer complementary transition

  5. Antitubercular and fluorescence studies of copper(II) complexes with quinolone family member, ciprofloxacin

    NASA Astrophysics Data System (ADS)

    Kharadi, G. J.

    2011-09-01

    Four new mixed-ligand complexes of Cu(II) with ciprofloxacin (Cip) and uninegative bidentate ligands have been synthesized and characterized. The structure of mixed-ligand complexes was investigated using spectroscopic method, physicochemical and elemental analyses. The fluorescence spectra of complexes show red shift, which may be due to the chelation by the ligands to the metal ion. It enhances ligand ability to accept electrons and decreases the electron transition energy. Antimycobacterial screening of ligand and its copper compound against Mycobacterium tuberculosis shows clear enhancement in the antitubercular activity upon copper complexation.

  6. A new fullerene complexation ligand: N-pyridylfulleropyrrolidine.

    PubMed

    Tat, Fatma T; Zhou, Zhiguo; MacMahon, Shaun; Song, Fayi; Rheingold, Arnold L; Echegoyen, Luis; Schuster, David I; Wilson, Stephen R

    2004-07-01

    The subject of this paper is a new fullerene building block design with the potential for defined geometry and good electronic communication. The synthesis and characterization of a new pyridinofullerene ligand capable of forming axially symmetric complexes with metalloporphyrins is reported. X-ray structural and molecular modeling studies, (1)H NMR, UV-vis spectroscopy, electrochemistry studies, and fluorescence quenching data support the formation of a strong complex between the new ligand and the metal center of ZnTPP. On the basis of computational studies, the highest occupied molecular orbital (HOMO) of this ligand is significantly different from a model compound with insulating carbons between the pyridine and the fullerene. The N-pyridinium fulleropyrrolidine salts of the new ligand and model compound were also prepared and their spectral and electrochemical properties are reported. PMID:15230581

  7. Fluorescent refrigeration

    DOEpatents

    Epstein, R.I.; Edwards, B.C.; Buchwald, M.I.; Gosnell, T.R.

    1995-09-05

    Fluorescent refrigeration is based on selective radiative pumping, using substantially monochromatic radiation, of quantum excitations which are then endothermically redistributed to higher energies. Ultimately, the populated energy levels radiatively deexcite emitting, on the average, more radiant energy than was initially absorbed. The material utilized to accomplish the cooling must have dimensions such that the exciting radiation is strongly absorbed, but the fluorescence may exit the material through a significantly smaller optical pathlength. Optical fibers and mirrored glasses and crystals provide this requirement. 6 figs.

  8. Fluorescence Microscopy

    PubMed Central

    Sanderson, Michael J.; Smith, Ian; Parker, Ian; Bootman, Martin D.

    2016-01-01

    Fluorescence microscopy is a major tool with which to monitor cell physiology. Although the concepts of fluorescence and its optical separation using filters remain similar, microscope design varies with the aim of increasing image contrast and spatial resolution. The basics of wide-field microscopy are outlined to emphasize the selection, advantages, and correct use of laser scanning confocal microscopy, two-photon microscopy, scanning disk confocal microscopy, total internal reflection, and super-resolution microscopy. In addition, the principles of how these microscopes form images are reviewed to appreciate their capabilities, limitations, and constraints for operation. PMID:25275114

  9. Surface ligands affect photoinduced modulation of the quantum dots optical performance

    NASA Astrophysics Data System (ADS)

    Krivenkov, Victor A.; Samokhvalov, Pavel S.; Linkov, Pavel A.; Solovyeva, Daria O.; Kotkovskii, Gennadii E.; Chistyakov, Alexander A.; Nabiev, Igor

    2014-05-01

    Changes of optical properties of the solutions of CdSe/ZnS quantum dots (QDs) covered with the trioctylphosphine oxide (TOPO) ligands under the pulsed ultraviolet (UV) laser irradiation are observed. The fluorescence quantum yield (QY) of QDs decreases by more than an order of magnitude when the radiation dose approaches 2 × 10-15 J per particle. This process is accompanied by a blue shift of both fluorescence and the first excitonic absorption peaks. The fluorescence quenching becomes less pronounced when the overall TOPO content in the solution is increased. When ТОРО ligands are replaced with n-hexadecylamine (HDA), QY and spectral properties are not changed at the same irradiation conditions. We assume that the above changes of the optical properties are associated with photooxidation of TOPO ligands by excited QD. Such process is less probable for the HDA ligand due to its different energy structure.

  10. Silent, fluorescent labeling of native neuronal receptors.

    PubMed

    Vytla, Devaiah; Combs-Bachmann, Rosamund E; Hussey, Amanda M; Hafez, Ismail; Chambers, James J

    2011-10-21

    We have developed a minimally-perturbing strategy that enables labeling and subcellular visualization of endogenous dendritic receptors on live, wild-type neurons. Specifically, calcium-permeable non-NMDA glutamate receptors expressed in hippocampal neurons can be targeted with this novel synthetic tri-functional molecule. This ligand-directed probe was targeted towards AMPA receptors and bears an electrophilic group for covalent bond formation with an amino acid side chain on the extracellular side of the ion channel. This molecule was designed in such a way that the use-dependent, polyamine-based ligand accumulates the chemically-reactive group at the extracellular side of these polyamine-sensitive receptors, thereby allowing covalent bond formation between an electrophilic moiety on the nanoprobe and a nucleophilic amino acid sidechain on the receptor. Bioconjugation of this molecule results in a stable covalent bond between the nanoprobe and the target receptor. Subsequent photolysis of a portion of the nanoprobe may then be employed to effect ligand release allowing the receptor to re-enter the non-liganded state, all the while retaining the fluorescent beacon for visualization. This technology allows for rapid fluorescent labeling of native polyamine-sensitive receptors and further advances the field of fluorescent labeling of native biological molecules. PMID:21897969

  11. LigandRNA: computational predictor of RNA–ligand interactions

    PubMed Central

    Philips, Anna; Milanowska, Kaja; Łach, Grzegorz; Bujnicki, Janusz M.

    2013-01-01

    RNA molecules have recently become attractive as potential drug targets due to the increased awareness of their importance in key biological processes. The increase of the number of experimentally determined RNA 3D structures enabled structure-based searches for small molecules that can specifically bind to defined sites in RNA molecules, thereby blocking or otherwise modulating their function. However, as of yet, computational methods for structure-based docking of small molecule ligands to RNA molecules are not as well established as analogous methods for protein-ligand docking. This motivated us to create LigandRNA, a scoring function for the prediction of RNA–small molecule interactions. Our method employs a grid-based algorithm and a knowledge-based potential derived from ligand-binding sites in the experimentally solved RNA–ligand complexes. As an input, LigandRNA takes an RNA receptor file and a file with ligand poses. As an output, it returns a ranking of the poses according to their score. The predictive power of LigandRNA favorably compares to five other publicly available methods. We found that the combination of LigandRNA and Dock6 into a “meta-predictor” leads to further improvement in the identification of near-native ligand poses. The LigandRNA program is available free of charge as a web server at http://ligandrna.genesilico.pl. PMID:24145824

  12. Analysis of macromolecules, ligands and macromolecule-ligand complexes

    DOEpatents

    Von Dreele, Robert B.

    2008-12-23

    A method for determining atomic level structures of macromolecule-ligand complexes through high-resolution powder diffraction analysis and a method for providing suitable microcrystalline powder for diffraction analysis are provided. In one embodiment, powder diffraction data is collected from samples of polycrystalline macromolecule and macromolecule-ligand complex and the refined structure of the macromolecule is used as an approximate model for a combined Rietveld and stereochemical restraint refinement of the macromolecule-ligand complex. A difference Fourier map is calculated and the ligand position and points of interaction between the atoms of the macromolecule and the atoms of the ligand can be deduced and visualized. A suitable polycrystalline sample of macromolecule-ligand complex can be produced by physically agitating a mixture of lyophilized macromolecule, ligand and a solvent.

  13. EGF receptor ligands: recent advances

    PubMed Central

    Singh, Bhuminder; Carpenter, Graham; Coffey, Robert J.

    2016-01-01

    Seven ligands bind to and activate the mammalian epidermal growth factor (EGF) receptor (EGFR/ERBB1/HER1): EGF, transforming growth factor-alpha (TGFA), heparin-binding EGF-like growth factor (HBEGF), betacellulin (BTC), amphiregulin (AREG), epiregulin (EREG), and epigen (EPGN). Of these, EGF, TGFA, HBEGF, and BTC are thought to be high-affinity ligands, whereas AREG, EREG, and EPGN constitute low-affinity ligands. This focused review is meant to highlight recent studies related to actions of the individual EGFR ligands, the interesting biology that has been uncovered, and relevant advances related to ligand interactions with the EGFR.

  14. Protein chip analysis by probing time-resolved UV fluorescence

    NASA Astrophysics Data System (ADS)

    Grigaravicius, Paulius; Dietrich, Rüdiger; Fritzsche, Wolfgang; Greulich, Karl Otto; Horn, Uwe; Knoll, Dietmar; Peters, Sven; Striebel, Hans-Martin; Schellenberg, Peter

    2007-07-01

    We describe a novel label-free method to analyse protein interactions on microarrays as well as in solution. By this technique the time resolved native protein fluorescence in the UV is probed. The method is based on alterations of the protein upon ligand binding, and, as a consequence, of alterations of the environment of the proteins' aromatic amino acids. These amino acids act as internal probes, and as a result, the fluorescence lifetime of the proteins change due to binding to a ligand partner such as another protein. We were able to demonstrate the feasibility of the method with many compounds, including protein-protein, protein-antibody, protein-nucleic acid and protein-small ligand pairs. Unlike to many other label-free techniques, the sensitivity of the method does not depend on the size of the counterbinding ligand and therefore is particularly suitable for drug monitoring, when small molecules are involved.

  15. Porphyrin-based design of bioinspired multitarget quadruplex ligands.

    PubMed

    Laguerre, Aurélien; Desbois, Nicolas; Stefan, Loic; Richard, Philippe; Gros, Claude P; Monchaud, David

    2014-09-01

    Secondary nucleic acid structures, such as DNA and RNA quadruplexes, are potential targets for cancer therapies. Ligands that interact with these targets could thus find application as anticancer agents. Synthetic G-quartets have recently found numerous applications, including use as bioinspired G-quadruplex ligands. Herein, the design, synthesis and preliminary biophysical evaluation of a new prototype multitarget G-quadruplex ligand, (PNA)PorphySQ, are reported, where peptidic nucleic acid guanine ((PNA)G) was incorporated in the porphyrin-templated synthetic G-quartet (PorphySQ). Using fluorescence resonance energy transfer (FRET)-melting experiments, PorphySQ was shown to possess enhanced quadruplex-interacting properties thanks to the presence of four positively charged (PNA)G residues that improve its electrostatic interactions with the binding site of both DNA and RNA quadruplexes (i.e., their negatively charged and accessible G-quartets), thereby making (PNA)PorphySQ an interesting prototype of a multitarget ligand. Both the chemical stability and water solubility of (PNA)PorphySQ are improved over the non-PNA derivative (PorphySQ), which are desirable properties for drug development, and while improvements remain to be made, this ligand is a promising lead for the further development of multitarget G-quadruplex ligands. PMID:24678052

  16. Ligand-Dependent Conformational Dynamics of Dihydrofolate Reductase

    PubMed Central

    Reddish, Michael J.; Vaughn, Morgan B.; Fu, Rong; Dyer, R. Brian

    2016-01-01

    Enzymes are known to change among several conformational states during turnover. The role of such dynamic structural changes in catalysis is not fully understood. The influence of dynamics in catalysis can be inferred, but not proven, by comparison of equilibrium structures of protein variants and protein–ligand complexes. A more direct way to establish connections between protein dynamics and the catalytic cycle is to probe the kinetics of specific protein motions in comparison to progress along the reaction coordinate. We have examined the enzyme model system dihydrofolate reductase (DHFR) from Escherichia coli with tryptophan fluorescence-probed temperature-jump spectroscopy. We aimed to observe the kinetics of the ligand binding and ligand-induced conformational changes of three DHFR complexes to establish the relationship among these catalytic steps. Surprisingly, in all three complexes, the observed kinetics do not match a simple sequential two-step process. Through analysis of the relationship between ligand concentration and observed rate, we conclude that the observed kinetics correspond to the ligand binding step of the reaction and a noncoupled enzyme conformational change. The kinetics of the conformational change vary with the ligand's identity and presence but do not appear to be directly related to progress along the reaction coordinate. These results emphasize the need for kinetic studies of DHFR with highly specific spectroscopic probes to determine which dynamic events are coupled to the catalytic cycle and which are not. PMID:26901612

  17. Semiquantitative fluorescence method for bioconjugation analysis.

    PubMed

    Brasil, Aluízio G; Carvalho, Kilmara H G; Leite, Elisa S; Fontes, Adriana; Santos, Beate Saegesser

    2014-01-01

    Quantum dots (QDs) have been used as fluorescent probes in biological and medical fields such as bioimaging, bioanalytical, and immunofluorescence assays. For these applications, it is important to characterize the QD-protein bioconjugates. This chapter provides details on a versatile method to confirm quantum dot-protein conjugation including the required materials and instrumentation in order to perform the step-by-step semiquantitative analysis of the bioconjugation efficiency by using fluorescence plate readings. Although the protocols to confirm the QD-protein attachment shown here were developed for CdTe QDs coated with specific ligands and proteins, the principles are the same for other QDs-protein bioconjugates. PMID:25103803

  18. Bright Fluorescence Monitoring System Utilizing Zoanthus sp. Green Fluorescent Protein (ZsGreen) for Human G-Protein-Coupled Receptor Signaling in Microbial Yeast Cells

    PubMed Central

    Nakamura, Yasuyuki; Ishii, Jun; Kondo, Akihiko

    2013-01-01

    G-protein-coupled receptors (GPCRs) are currently the most important pharmaceutical targets for drug discovery because they regulate a wide variety of physiological processes. Consequently, simple and convenient detection systems for ligands that regulate the function of GPCR have attracted attention as powerful tools for new drug development. We previously developed a yeast-based fluorescence reporter ligand detection system using flow cytometry. However, using this conventional detection system, fluorescence from a cell expressing GFP and responding to a ligand is weak, making detection of these cells by fluorescence microscopy difficult. We here report improvements to the conventional yeast fluorescence reporter assay system resulting in the development of a new highly-sensitive fluorescence reporter assay system with extremely bright fluorescence and high signal-to-noise (S/N) ratio. This new system allowed the easy detection of GPCR signaling in yeast using fluorescence microscopy. Somatostatin receptor and neurotensin receptor (implicated in Alzheimer’s disease and Parkinson’s disease, respectively) were chosen as human GPCR(s). The facile detection of binding to these receptors by cognate peptide ligands was demonstrated. In addition, we established a highly sensitive ligand detection system using yeast cell surface display technology that is applicable to peptide screening, and demonstrate that the display of various peptide analogs of neurotensin can activate signaling through the neurotensin receptor in yeast cells. Our system could be useful for identifying lead peptides with agonistic activity towards targeted human GPCR(s). PMID:24340008

  19. Ligand-gated Diffusion Across the Bacterial Outer Membrane

    SciTech Connect

    B Lepore; M Indic; H Pham; E Hearn; D Patel; B van den Berg

    2011-12-31

    Ligand-gated channels, in which a substrate transport pathway is formed as a result of the binding of a small-molecule chemical messenger, constitute a diverse class of membrane proteins with important functions in prokaryotic and eukaryotic organisms. Despite their widespread nature, no ligand-gated channels have yet been found within the outer membrane (OM) of Gram-negative bacteria. Here we show, using in vivo transport assays, intrinsic tryptophan fluorescence and X-ray crystallography, that high-affinity (submicromolar) substrate binding to the OM long-chain fatty acid transporter FadL from Escherichia coli causes conformational changes in the N terminus that open up a channel for substrate diffusion. The OM long-chain fatty acid transporter FadL from E. coli is a unique paradigm for OM diffusion-driven transport, in which ligand gating within a {beta}-barrel membrane protein is a prerequisite for channel formation.

  20. New fluorescence reactions in DNA cytochemistry. 2. Microscopic and spectroscopic studies on fluorescent aluminum complexes

    SciTech Connect

    Del Castillo, P.; Llorente, A.R.; Gomez, A.; Gosalvez, J.; Goyanes, V.J.; Stockert, J.C. )

    1990-02-01

    Metal-dye complexes are widely applied in light microscopic techniques for chromatin staining (e.g., hematoxylin and carmine), but fluorescent complexes between phosphate-binding cations and suitable ligands have been little used. Preformed and postformed Al complexes with different anionic dyes induced strong and selective fluorescence reactions in nuclei from chicken blood smears, frozen sections, paraffin-embedded sections and Epon-embedded sections of mouse and rat tissues, mitotic chromosomes, meiotic chromosomes and kinetoplasts of Trypanosoma cruzi epimastigotes. The DNA-dependent fluorescence of these structures showed a very low fading rate. The emission colors were related to the ligand. The most suitable compounds for forming fluorescent Al chelates were 8-hydroxyquinoline, morin, nuclear fast red and purpurin. Staining with diluted carmine solutions and InCl3 mordanting, followed by 8-hydroxyquinoline, also induced chromatin fluorescence. After treating isolated mouse chromosomes with the preformed complex Al-nuclear fast red, x-ray microanalysis indicated a P:Al:dye binding ratio of about 40:15:1. The selectivity, stability and easy formation of these fluorescent Al complexes are obvious advantages for their use as new cytochemical probes in cytologic studies.

  1. Scatchard analysis of fluorescent concanavalin A binding to lymphocytes

    SciTech Connect

    Gordon, I.L.

    1995-07-01

    Standard Scatchard analysis of ligand binding to cell receptors requires the use of isotopes and is imprecise at low ligand concentrations. To evaluate the feasibility of Scatchard analysis via fluorescence flow cytometry, the binding of fluorescein isothio-cyanate-derivatized concanavalin A (FITC-ConA) to murine lymphocytes at 4{degrees}C was compared to {sup 125}I-ConA binding. A FACS IV flow cytometer was used for analysis of cells after fluorescent ligand binding. A simple spectrophotometric technique was used to calibrate the relation between cytometer-determined fluorescence and ligand binding per cell. As FITC-ConA binding showed a quasi-Gaussian distribution, the mean number of molecules bound per cell was easily calculated. Scatchard analysis of FITC-ConA binding yielded results (1.9 x 10{sup 6} receptors/cell, K = 3.6 x 10{sup -15}) similar to those obtained With {sup 125}I-ConA (1.4 x 10{sup 6} receptors/cell, K = 5.2 x 10{sup -15}). Cytometric Scatchard plots showed less scatter and seemed more precise, suggesting superiority to radioactive ligand measurements, particularly at low ligand concentrations. 32 refs., 4 figs., 1 tab.

  2. Glucose sensing molecules having selected fluorescent properties

    DOEpatents

    Satcher, Jr., Joe H.; Lane, Stephen M.; Darrow, Christopher B.; Cary, Douglas R.; Tran, Joe Anh

    2004-01-27

    An analyte sensing fluorescent molecule that employs intramolecular electron transfer is designed to exhibit selected fluorescent properties in the presence of analytes such as saccharides. The selected fluorescent properties include excitation wavelength, emission wavelength, fluorescence lifetime, quantum yield, photostability, solubility, and temperature or pH sensitivity. The compound comprises an aryl or a substituted phenyl boronic acid that acts as a substrate recognition component, a fluorescence switch component, and a fluorophore. The fluorophore and switch component are selected such that the value of the free energy for electron transfer is less than about 3.0 kcal mol.sup.-1. Fluorescent compounds are described that are excited at wavelengths greater than 400 nm and emit at wavelengths greater than 450 nm, which is advantageous for optical transmission through skin. The fluorophore is typically selected from transition metal-ligand complexes and thiazine, oxazine, oxazone, or oxazine-one as well as anthracene compounds. The fluorescent compound can be immobilized in a glucose permeable biocompatible polymer matrix that is implantable below the skin.

  3. Recent conjugation strategies of small organic fluorophores and ligands for cancer-specific bioimaging.

    PubMed

    Ha, Yonghwang; Choi, Hyun-Kyung

    2016-03-25

    Conjugation between various small fluorophores and specific ligands has become one of the main strategies for bioimaging in disease diagnosis, medicinal chemistry, immunology, and fluorescence-guided surgery, etc. Herein, we present our review of recent studies relating to molecular fluorescent imaging techniques for various cancers in cell-based and animal-based models. Various organic fluorophores, especially near-infrared (NIR) probes, have been employed with specific ligands. Types of ligands used were small molecules, peptides, antibodies, and aptamers; each has specific affinities for cellular receptor proteins, cancer-specific antigens, enzymes, and nucleic acids. This review can aid in the selection of cancer-specific ligands and fluorophores, and may inspire the further development of new conjugation strategies in various cellular and animal models. PMID:26892219

  4. Fluorescence of dental porcelain.

    PubMed

    Monsénégo, G; Burdairon, G; Clerjaud, B

    1993-01-01

    This study of the fluorescence of natural enamel and of dental ceramics shows the fluorescence of ceramics not containing rare earths decreases when the color saturation increases; the fluorescence of samples of the same shade guide are not homogenous; some guides show a strong green fluorescence; and two shade guides of the same origin can present completely different fluorescence. The cementing medium can affect the fluorescence of a ceramic prosthesis. PMID:8455155

  5. Monitoring ligand-mediated internalization of G protein-coupled receptor as a novel pharmacological approach.

    PubMed

    Fukunaga, Shin'ichi; Setoguchi, Shingo; Hirasawa, Akira; Tsujimoto, Gozoh

    2006-12-01

    Agonist activation of a G protein-coupled receptor (GPCR) results in the redistribution of the receptor protein away from the cell surface into internal cellular compartments through a process of endocytosis known as internalization. Visualization of receptor internalization has become experimentally practicable by using fluorescent reagents such as green fluorescent protein (GFP). In this study, we examined whether the ligand-mediated internalization of a GPCR can be exploited for pharmacological evaluations. We acquired fluorescent images of cells expressing GFP-labeled GPCRs and evaluated the ligand-mediated internalization quantitatively by image processing. Using beta2-adrenoceptor and vasopressin V1a receptor as model GPCRs that couple to Gs and Gq, respectively, we first examined whether these GFP-tagged GPCRs exhibited appropriate pharmacology. The rank order of receptor internalization potency for a variety of agonists and antagonists specific to each receptor corresponded well with that previously observed in ligand binding studies. In addition to chemical ligand-induced internalization, this cell-based fluorescence imaging system successfully monitored the internalization of the proton-sensing GPCR TDAG8, and that of the free fatty acid-sensitive GPCR GPR120. The results show that monitoring receptor internalization can be a useful approach for pharmacological characterization of GPCRs and in fishing for ligands of orphan GPCRs. PMID:16978657

  6. Molecular studies of pH dependent ligand interactions with the low-density lipoprotein receptor*

    PubMed Central

    Yamamoto, Taichi; Chen, Hsuan-Chih; Guigard, Emmanuel; Kay, Cyril M.; Ryan, Robert O.

    2009-01-01

    Ligand release from the low-density lipoprotein receptor (LDLR) has been postulated to involve a “histidine switch” induced intra-molecular rearrangement that discharges bound ligand. A recombinant soluble low-density lipoprotein receptor (sLDLR) was employed in ligand binding experiments with a fluorescent-tagged variant apolipoprotein E-N-terminal domain (apoE-NT). Binding was monitored as a function of fluorescence resonance energy transfer (FRET) from excited Trp residues in sLDLR to an extrinsic fluorophore covalently attached to Trp null apoE3-NT. In binding experiments with wild type (WT) sLDLR, FRET-dependent AEDANS fluorescence decreased as the pH was lowered. To investigate the role of His190, His562 and His586 in sLDLR on pH dependent ligand binding and discharge, site directed mutagenesis studies were performed. Compared to WT sLDLR, triple His→Ala mutant sLDLR displayed attenuated pH-dependent ligand binding and decreased ligand release as a function of low pH. When these His residues were substituted for Lys, whose positively charged side chain does not ionize over this pH range, ligand binding was nearly abolished at all pH values. When sequential His to Lys mutants were examined, evidence obtained suggested that His562 and His586 function cooperatively. Whereas the sedimentation coefficient for WT sLDLR increased upon lowering the pH from 7 to 5, no such change occurred in the case of the triple Lys mutant receptor or a His562Lys / His586Lys double mutant receptor. The data support the existence of a cryptic, histidine side chain ionization-dependent alternative ligand that modulates ligand discharge via conformational reorganization. PMID:18847225

  7. Fluorescent or not? Size-dependent fluorescence switching for polymer-stabilized gold clusters in the 1.1-1.7 nm size range.

    PubMed

    Schaeffer, Nicolas; Tan, Bien; Dickinson, Calum; Rosseinsky, Matthew J; Laromaine, Anna; McComb, David W; Stevens, Molly M; Wang, Yiqian; Petit, Laure; Barentin, Catherine; Spiller, David G; Cooper, Andrew I; Lévy, Raphaël

    2008-09-14

    The synthesis of fluorescent water-soluble gold nanoparticles by the reduction of a gold salt in the presence of a designed polymer ligand is described, the size and fluorescence of the particles being controlled by the polymer to gold ratio; the most fluorescent nanomaterial has a 3% quantum yield, a 1.1 nm gold core and a 6.9 nm hydrodynamic radius. PMID:18758601

  8. Galanin Receptors and Ligands

    PubMed Central

    Webling, Kristin E. B.; Runesson, Johan; Bartfai, Tamas; Langel, Ülo

    2012-01-01

    The neuropeptide galanin was first discovered 30 years ago. Today, the galanin family consists of galanin, galanin-like peptide (GALP), galanin-message associated peptide (GMAP), and alarin and this family has been shown to be involved in a wide variety of biological and pathological functions. The effect is mediated through three GPCR subtypes, GalR1-3. The limited number of specific ligands to the galanin receptor subtypes has hindered the understanding of the individual effects of each receptor subtype. This review aims to summarize the current data of the importance of the galanin receptor subtypes and receptor subtype specific agonists and antagonists and their involvement in different biological and pathological functions. PMID:23233848

  9. G-quadruplex DNA bound by a synthetic ligand is highly dynamic.

    PubMed

    Jena, Prakrit V; Shirude, Pravin S; Okumus, Burak; Laxmi-Reddy, Katta; Godde, Frédéric; Huc, Ivan; Balasubramanian, Shankar; Ha, Taekjip

    2009-09-01

    Using single molecule fluorescence resonance energy transfer, we investigated the interaction between a quadruplex-binding ligand and the human telomeric G-quadruplex. The binding of quinolinecarboxamide macrocycle to telomeric DNA was essentially irreversible and selectively induced and favored one quadruplex conformation. The ligand-quadruplex complex displayed intramolecular dynamics including quadruplex folding and unfolding in the absence of ligand association and dissociation. We report that the G-quadruplex can be stabilized without preventing the intrinsic intramolecular dynamics of telomeric DNA. PMID:19685880

  10. Effect of surface ligands on the optical properties of aqueous soluble CdTe quantum dots

    PubMed Central

    2012-01-01

    We investigate systematically the influence of the nature of thiol-type capping ligands on the optical and structural properties of highly luminescent CdTe quantum dots synthesized in aqueous media, comparing mercaptopropionic acid (MPA), thioglycolic acid (TGA), 1-thioglycerol (TGH), and glutathione (GSH). The growth rate, size distribution, and quantum yield strongly depend on the type of surface ligand used. While TGH binds too strongly to the nanocrystal surface inhibiting growth, the use of GSH results in the fastest growth kinetics. TGA and MPA show intermediate growth kinetics, but MPA yields a much lower initial size distribution than TGA. The obtained fluorescence quantum yields range from 38% to 73%. XPS studies unambiguously put into evidence the formation of a CdS shell on the CdTe core due to the thermal decomposition of the capping ligands. This shell is thicker when GSH is used as ligand, as compared with TGA ligands. PMID:23017183

  11. Complexation of trivalent americium and lanthanides with terdentate 'N' donor ligands: the role of rigidity in the ligand structure.

    PubMed

    Bhattacharyya, Arunasis; Gadly, Trilochan; Pathak, Priyanath; Ghosh, Sunil K; Mohapatra, Manoj; Ghanty, Tapan K; Mohapatra, Prasanta K

    2014-08-28

    A systematic study on the Ln(3+) complexation behaviour with two terdentate 'N' donor ligands of varying structural rigidity, viz. 5,6-dimethyl-(1,2,4)-triazinylbipyridine (Me2TBipy) and 5,6-dimethyl-(1,2,4)-triazinylphenanthroline (Me2TPhen), is performed in the present work by UV-Vis spectrophotometry, time resolved fluorescence spectroscopy (TRFS) and electrospray ionization mass spectrometric (ESI-MS) studies. These studies indicate the formation of a 1 : 1 complex of La(3+), 1 : 2 complexes of Eu(3+) and Er(3+) with both the ligands. Density functional theoretical (DFT) study is carried out to determine the solution phase structure of the Eu(3+) complex considering the species (from UV-Vis spectrophotometry) and C2v site symmetry around the Eu(3+) ion (from TRFS study). Me2TPhen is found to be a stronger complexing ligand as compared to Me2TBipy irrespective of the Ln(3+) ions. The solid state crystal structure of the La(3+) complex of Me2TPhen is determined using the single crystal X-ray diffraction (SCXRD) technique. The complexation of the trivalent Am(3+) ion is also studied with both these ligands using UV-Vis spectrophotometric titrations which show the formation of 1 : 2 complexes with higher complexation constant values as compared to all the Ln(3+) ions studied, indicating the selectivity of these ligands for the trivalent actinides over the lanthanides. PMID:25001925

  12. Cellular trafficking of quantum dot-ligand bioconjugates and their induction of changes in normal routing of unconjugated ligands.

    PubMed

    Tekle, Christina; Deurs, Bo van; Sandvig, Kirsten; Iversen, Tore-Geir

    2008-07-01

    Can quantum dots (Qdots) act as relevant intracellular probes to investigate routing of ligands in live cells? The intracellular trafficking of Qdots that were coupled to the plant toxin ricin, Shiga toxin, or the ligand transferrin (Tf) was studied by confocal fluorescence microscopy. The Tf:Qdots were internalized by clathrin-dependent endocytosis as fast as Tf, but their recycling was blocked. Unlike Shiga toxin, the Shiga:Qdot bioconjugate was not routed to the Golgi apparatus. The internalized ricin:Qdot bioconjugates localized to the same endosomes as ricin itself but could not be visualized in the Golgi apparatus. Importantly, we find that the endosomal accumulation of ricin:Qdots affects endosome-to-Golgi transport of both ricin and Shiga toxin: Transport of ricin was reduced whereas transport of Shiga toxin was increased. In conclusion, the data reveal that, although coupling of Qdots to a ligand does not necessarily change the endocytic pathway normally used by the ligands studied, it appears that the ligand-coupled Qdot nanoparticles can be arrested within endosomes and somehow perturb the normal endosomal sorting in cells. Thus, the results demonstrate that Qdots may have severe consequences on cell physiology. PMID:18570482

  13. Bifunctional DTPA-type ligand

    SciTech Connect

    Gansow, O.A.; Brechbiel, M.W.

    1990-03-26

    The subject matter of the invention relates to bifunctional cyclohexyl DTPA ligands and methods of using these compounds. Specifically, such ligands are useful for radiolabeling proteins with radioactive metals, and can consequently be utilized with respect to radioimmunoimaging and/or radioimmunotherapy.

  14. Al(+)-ligand binding energies

    NASA Technical Reports Server (NTRS)

    Sodupe, M.; Bauschlicher, Charles W., Jr.

    1991-01-01

    Ab initio calculations are used to optimize the structure and determine the binding energies of Al(+) to a series of ligands. For Al(+)-CN, the bonding was found to have a large covalent component. For the remaining ligands, the bonding is shown to be electrostatic in origin. The results obtained for Al(+) are compared with those previously reported for Mg(+).

  15. Organometallic Ru(II) Photosensitizers Derived from π-Expansive Cyclometalating Ligands: Surprising Theranostic PDT Effects.

    PubMed

    Sainuddin, Tariq; McCain, Julia; Pinto, Mitch; Yin, Huimin; Gibson, Jordan; Hetu, Marc; McFarland, Sherri A

    2016-01-01

    The purpose of the present study was to investigate the influence of π-expansive cyclometalating ligands on the photophysical and photobiological properties of organometallic Ru(II) compounds. Four compounds with increasing π conjugation on the cyclometalating ligand were prepared, and their structures were confirmed by HPLC, 1D and 2D (1)H NMR, and mass spectrometry. The properties of these compounds differed substantially from their Ru(II) polypyridyl counterparts. Namely, they were characterized by red-shifted absorption, very weak to no room temperature phosphorescence, extremely short phosphorescence state lifetimes (<10 ns), low singlet oxygen quantum yields (0.5-8%), and efficient ligand-centered fluorescence. Three of the metal complexes were very cytotoxic to cancer cells in the dark (EC50 values = 1-2 μM), in agreement with what has traditionally been observed for Ru(II) compounds derived from small C^N ligands. Surprisingly, the complex derived from the most π-expansive cyclometalating ligand exhibited no cytotoxicity in the dark (EC50 > 300 μM) but was phototoxic to cells in the nanomolar regime. Exceptionally large phototherapeutic margins, exceeding 3 orders of magnitude in some cases, were accompanied by bright ligand-centered intracellular fluorescence in cancer cells. Thus, Ru(II) organometallic systems derived from π-expansive cyclometalating ligands, such 4,9,16-triazadibenzo[a,c]napthacene (pbpn), represent the first class of potent light-responsive Ru(II) cyclometalating agents with theranostic potential. PMID:26672769

  16. Natural ligand binding and transfer from liver fatty acid binding protein (LFABP) to membranes.

    PubMed

    De Gerónimo, Eduardo; Hagan, Robert M; Wilton, David C; Córsico, Betina

    2010-09-01

    Liver fatty acid-binding protein (LFABP) is distinctive among fatty acid-binding proteins because it binds more than one molecule of long-chain fatty acid and a variety of diverse ligands. Also, the transfer of fluorescent fatty acid analogues to model membranes under physiological ionic strength follows a different mechanism compared to most of the members of this family of intracellular lipid binding proteins. Tryptophan insertion mutants sensitive to ligand binding have allowed us to directly measure the binding affinity, ligand partitioning and transfer to model membranes of natural ligands. Binding of fatty acids shows a cooperative mechanism, while acyl-CoAs binding presents a hyperbolic behavior. Saturated fatty acids seem to have a stronger partition to protein vs. membranes, compared to unsaturated fatty acids. Natural ligand transfer rates are more than 200-fold higher compared to fluorescently-labeled analogues. Interestingly, oleoyl-CoA presents a markedly different transfer behavior compared to the rest of the ligands tested, probably indicating the possibility of specific targeting of ligands to different metabolic fates. PMID:20541621

  17. The maximal affinity of ligands

    PubMed Central

    Kuntz, I. D.; Chen, K.; Sharp, K. A.; Kollman, P. A.

    1999-01-01

    We explore the question of what are the best ligands for macromolecular targets. A survey of experimental data on a large number of the strongest-binding ligands indicates that the free energy of binding increases with the number of nonhydrogen atoms with an initial slope of ≈−1.5 kcal/mol (1 cal = 4.18 J) per atom. For ligands that contain more than 15 nonhydrogen atoms, the free energy of binding increases very little with relative molecular mass. This nonlinearity is largely ascribed to nonthermodynamic factors. An analysis of the dominant interactions suggests that van der Waals interactions and hydrophobic effects provide a reasonable basis for understanding binding affinities across the entire set of ligands. Interesting outliers that bind unusually strongly on a per atom basis include metal ions, covalently attached ligands, and a few well known complexes such as biotin–avidin. PMID:10468550

  18. Detection of boronic acid derivatives in cells using a fluorescent sensor.

    PubMed

    Hattori, Yoshihide; Ishimura, Miki; Ohta, Youichirou; Takenaka, Hiroshi; Watanabe, Tsubasa; Tanaka, Hiroki; Ono, Koji; Kirihata, Mitsunori

    2015-07-01

    The detection of boron-containing compounds requires very expensive facilities and/or tedious sample pretreatments. In an effort to develop a convenient detection method for boronic acid derivatives, boron chelating-ligands were synthesized for use as fluorescent sensors. In this paper, the synthesis and properties of fluorescent sensors for boronic acid derivatives are reported. PMID:26022725

  19. Predicting target-ligand interactions using protein ligand-binding site and ligand substructures

    PubMed Central

    2015-01-01

    Background Cell proliferation, differentiation, Gene expression, metabolism, immunization and signal transduction require the participation of ligands and targets. It is a great challenge to identify rules governing molecular recognition between chemical topological substructures of ligands and the binding sites of the targets. Methods We suppose that the ligand-target interactions are determined by ligand substructures as well as the physical-chemical properties of the binding sites. Therefore, we propose a fragment interaction model (FIM) to describe the interactions between ligands and targets, with the purpose of facilitating the chemical interpretation of ligand-target binding. First we extract target-ligand complexes from sc-PDB database, based on which, we get the target binding sites and the ligands. Then we represent each binding site as a fragment vector based on a target fragment dictionary that is composed of 199 clusters (denoted as fragements in this work) obtained by clustering 4200 trimers according to their physical-chemical properties. And then, we represent each ligand as a substructure vector based on a dictionary containing 747 substructures. Finally, we build the FIM by generating the interaction matrix M (representing the fragment interaction network), and the FIM can later be used for predicting unknown ligand-target interactions as well as providing the binding details of the interactions. Results The five-fold cross validation results show that the proposed model can get higher AUC score (92%) than three prevalence algorithms CS-PD (80%), BLM-NII (85%) and RF (85%), demonstrating the remarkable predictive ability of FIM. We also show that the ligand binding sites (local information) overweight the sequence similarities (global information) in ligand-target binding, and introducing too much global information would be harmful to the predictive ability. Moreover, The derived fragment interaction network can provide the chemical insights on

  20. Fundamentals of fluorescence and fluorescence microscopy.

    PubMed

    Wolf, David E

    2013-01-01

    This chapter discusses the fundamental physics of fluorescence. The application of fluorescence to microscopy represents an important transition in the development of microscopy, particularly as it applies to biology. It enables quantitating the amounts of specific molecules within a cell, determining whether molecules are complexing on a molecular level, measuring changes in ionic concentrations within cells and organelles, and measuring molecular dynamics. This chapter also discusses the issues important to quantitative measurement of fluorescence and focuses on four of quantitative measurements of fluorescence--boxcar-gated detection, streak cameras, photon correlation, and phase modulation. Although quantitative measurement presents many pitfalls to the beginner, it also presents significant opportunities to one skilled in the art. This chapter also examines how fluorescence is measured in the steady state and time domain and how fluorescence is applied in the modern epifluorescence microscope. PMID:23931503

  1. Micromolding of a Highly Fluorescent Reticular Coordination Polymer: Solvent-Mediated Reconfigurable Polymerization in a Soft Lithographic Mold

    SciTech Connect

    Y You; H Yang; J Chung; J Kim; Y Jung; S Park

    2011-12-31

    Coordination polymerization of pyridine-based ligands and zinc or silver ions was controlled by soft lithographic micromolding in capillaries. The polymer patterns that are produced are highly fluorescent and supramolecularly structured.

  2. [The fluorescence of terbium complex and its application in wavelength conversion membrane].

    PubMed

    Wang, Man-Li; Zhang, Xiao-Hui; Yin, Hong-Zong; Xu, Kun

    2013-04-01

    Terbium was selected as test material for its strong fluorescence effect, and sulfosalicylic acid was used as first ligand, polyvinyl alcohol and polyethylene glycol 2000 as co-ligand, the fluorescence property of complexes in the two systems of ethanol solution and aqueous solution was explored. It was obtained that the polyvinyl alcohol and polyethylene glycol 2000 are the excellent co-ligands. Further study showed that sufactant is good for fluorescence enhancement of the different complexes and especially sodium dodecyl sulfate is best while exploring the impact of acidity on the fluorescence intensity. Terbium-sulfosalicylic acid-polyvinyl alcohol complex was obtained under the conditions of 342 nm for excitation wavelength, and 545 nm for emission wavelength. Mixing the complex into the plastic film in proper proportion, the authors prepared the rare earth light conversion membrane which allowed ultraviolet portion of sunlight to convert to green light the crop photosythesis needed to effectively improve the photosynthetic efficiency. PMID:23841418

  3. Dithiocarbamates as capping ligands for water-soluble quantum dots.

    PubMed

    Zhang, Yanjie; Schnoes, Allison M; Clapp, Aaron R

    2010-11-01

    We investigated the suitability of dithiocarbamate (DTC) species as capping ligands for colloidal CdSe-ZnS quantum dots (QDs). DTC ligands are generated by reacting carbon disulfide (CS(2)) with primary or secondary amines on appropriate precursor molecules. A biphasic exchange procedure efficiently replaces the existing hydrophobic capping ligands on the QD surface with the newly formed DTCs. The reaction conversion is conveniently monitored by UV-vis absorption spectroscopy. Due to their inherent water solubility and variety of side chain functional groups, we used several amino acids as precursors in this reaction/exchange procedure. The performance of DTC-ligands, as evaluated by the preservation of luminescence and colloidal stability, varied widely among amino precursors. For the best DTC-ligand and QD combinations, the quantum yield of the water-soluble QDs rivaled that of the original hydrophobic-capped QDs dispersed in organic solvents. The mean density of DTC-ligands per nanocrystal was estimated through a mass balance calculation which suggested nearly complete coverage of the available nanocrystal surface. The accessibility of the QD surface was evaluated by self-assembly of His-tagged dye-labeled proteins and peptides using fluorescence resonance energy transfer. DTC-capped QDs were also exposed to cell cultures to evaluate their stability and potential use for biological applications. In general, DTC-capped CdSe-ZnS QDs have many advantages over other water-soluble QD formulations and provide a flexible chemistry for controlling the QD surface functionalization. Despite previous literature reports of DTC-stabilized nanocrystals, this study is the first formal investigation of a biphasic exchange method for generating biocompatible core-shell QDs. PMID:21053924

  4. Multidentate polymeric ligands for long-term bioimaging using highly stable and functionalized quantum dots

    NASA Astrophysics Data System (ADS)

    Giovanelli, Emerson; Muro, Eleonora; Tasso, Mariana; Sitbon, Gary; Hanafi, Mohamed; Pons, Thomas; Dubertret, Benoît.; Lequeux, Nicolas

    2014-03-01

    Colloidal fluorescent semiconductor nanocrystals, named "quantum dots", possess unique features, such as a tunable peak wavelength (according to their composition and their size) or a large absorption cross-section, that make them very attractive for biomedical imaging. Nevertheless, typical syntheses provide nanoparticles capped with hydrophobic ligands. To be used in long-term bioexperiments, they have thus to be modified to exhibit essentially a high colloidal stability in aqueous conditions, but also a low non-specific adsorption, a small size and functionalization moities. As all of these properties are controlled by the layer of coating ligands, we designed a bidentate monozwitterionic ligand, to first address the need of small-sized and antibiofouling hydrophilic probes. But the corresponding quantum dots revealed to be unstable in highly diluted conditions and difficult to functionalize. To further increase the affinity between the nanoparticles and their surrounding ligands, we synthesized a multidentate polyzwitterionic ligand, issued from the copolymerization of a bidentate monomer and a monozwitterionic one. The nanocrystals passivated by this polymeric ligand showed an exceptional colloidal stability, regardless of the medium conditions (pH, salinity, dilution, and biological environment), and we demonstrated the affinity of the polymer exceeded by three orders of magnitude that of the bidentate ligand. The synthesis of the multidentate polyzwitterionic ligand proved also to be easily tunable and allowed the facile introduction of reacting moieties. Further functionalization of the corresponding quantum dots with biomolecules led to successful specific targeting, which could be confirmed, as an example, through FRET experiments.

  5. Petasis-Ugi ligands: New affinity tools for the enrichment of phosphorylated peptides.

    PubMed

    Batalha, Íris L; Roque, Ana C A

    2016-09-15

    Affinity chromatography is a widespread technique for the enrichment and isolation of biologics, which relies on the selective and reversible interaction between affinity ligands and target molecules. Small synthetic affinity ligands are valuable alternatives due to their robustness, low cost and fast ligand development. This work reports, for the first time, the use of a sequential Petasis-Ugi multicomponent reaction to generate rationally designed solid-phase combinatorial libraries of small synthetic ligands, which can be screened for the selection of new affinity adsorbents towards biological targets. As a proof of concept, the Petasis-Ugi reaction was here employed in the discovery of affinity ligands suitable for phosphopeptide enrichment. A combinatorial library of 84 ligands was designed, synthesized on a chromatographic solid support and screened in situ for the specific binding of phosphopeptides binding human BRCA1C-terminal domains. The success of the reaction on the chromatographic matrix was confirmed by both inductively coupled plasma atomic emission spectroscopy and fluorescence microscopy. Three lead ligands were identified due to their superior performance in terms of binding capacity and selectivity towards the phosphorylated moiety on peptides, which showed the feasibility of the Petasis-Ugi reaction for affinity ligand development. PMID:27469904

  6. Ligand Identification Scoring Algorithm (LISA)

    PubMed Central

    Zheng, Zheng; Merz, Kenneth M.

    2011-01-01

    A central problem in de novo drug design is determining the binding affinity of a ligand with a receptor. A new scoring algorithm is presented that estimates the binding affinity of a protein-ligand complex given a three-dimensional structure. The method, LISA (Ligand Identification Scoring Algorithm), uses an empirical scoring function to describe the binding free energy. Interaction terms have been designed to account for van der Waals (VDW) contacts, hydrogen bonding, desolvation effects and metal chelation to model the dissociation equilibrium constants using a linear model. Atom types have been introduced to differentiate the parameters for VDW, H-bonding interactions and metal chelation between different atom pairs. A training set of 492 protein-ligand complexes was selected for the fitting process. Different test sets have been examined to evaluate its ability to predict experimentally measured binding affinities. By comparing with other well known scoring functions, the results show that LISA has advantages over many existing scoring functions in simulating protein-ligand binding affinity, especially metalloprotein-ligand binding affinity. Artificial Neural Network (ANN) was also used in order to demonstrate that the energy terms in LISA are well designed and do not require extra cross terms. PMID:21561101

  7. Proton-dependent zinc release from intracellular ligands

    PubMed Central

    Kiedrowski, Lech

    2014-01-01

    In cultured cortical and hippocampal neurons when intracellular pH drops from 6.6 to 6.1, yet unclear intracellular stores release micromolar amounts of Zn2+ into the cytosol. Mitochondria, acidic organelles, and/or intracellular ligands could release this Zn2+. Although exposure to the protonophore FCCP precludes re-loading of the mitochondria and acidic organelles with Zn2+, FCCP failed to compromise the ability of the intracellular stores to repeatedly release Zn2+. Therefore, Zn2+-releasing stores were not mitochondria or acidic organelles but rather intracellular Zn2+ ligands. To test which ligands might be involved, the rate of acid-induced Zn2+ release from complexes with cysteine, glutathione, histidine, aspartate, glutamate, glycine, and carnosine was investigated; [Zn2+] was monitored in vitro using the ratiometric Zn2+-sensitive fluorescent probe FuraZin-1. Carnosine failed to chelate Zn2+ but did chelate Cu2+; the remaining ligands chelated Zn2+ and upon acidification were releasing it into the medium. However, when pH was decreasing from 6.6 to 6.1, only zinc-cysteine complexes rapidly accelerated the rate of Zn2+ release. The zinc-cysteine complexes also released Zn2+ when a histidine-modifying agent, diethylpyrocarbonate, was applied at pH 7.2. Since the cytosolic zinc-cysteine complexes can contain micromolar amounts of Zn2+, these complexes may represent the stores responsible for an acid-induced intracellular Zn2+ release. PMID:24606401

  8. A model for the study of ligand binding to the ribosomal RNA helix h44

    SciTech Connect

    Dibrov, Sergey M.; Parsons, Jerod; Hermann, Thomas

    2010-09-02

    Oligonucleotide models of ribosomal RNA domains are powerful tools to study the binding and molecular recognition of antibiotics that interfere with bacterial translation. Techniques such as selective chemical modification, fluorescence labeling and mutations are cumbersome for the whole ribosome but readily applicable to model RNAs, which are readily crystallized and often give rise to higher resolution crystal structures suitable for detailed analysis of ligand-RNA interactions. Here, we have investigated the HX RNA construct which contains two adjacent ligand binding regions of helix h44 in 16S ribosomal RNA. High-resolution crystal structure analysis confirmed that the HX RNA is a faithful structural model of the ribosomal target. Solution studies showed that HX RNA carrying a fluorescent 2-aminopurine modification provides a model system that can be used to monitor ligand binding to both the ribosomal decoding site and, through an indirect effect, the hygromycin B interaction region.

  9. Novel ligands that target the mitochondrial membrane protein mitoNEET

    PubMed Central

    Bieganski, Robert M.; Yarmush, Martin L.

    2012-01-01

    Ligands of the thiazolidinedione (TZD) class of compounds, pioglitazone (Actos™) and rosiglitazone (Avandia™) are currently approved for treatment of type 2 diabetes and are known to bind to the PPAR-γ nuclear receptor subtype. Recent evidence suggesting PPAR-γ independent action of the TZDs led to the discovery of a novel integral outer mitochondrial membrane protein, mitoNEET. In spite of the several reported X-ray crystal structures of the unbound form of mitoNEET, the location and nature of the mitoNEET ligand binding sites (LBS) remain unknown. In this study, a molecular blind docking (BD) method was used to discover potential mitoNEET LBS and novel ligands, utilizing the program AutoDock Vina (v 1.0.2). Validation of BD was performed on the PPAR-γ receptor (PDB ID: 1ZGY) with the test compound rosiglitazone, demonstrating that the binding conformation of rosiglitazone determined by AutoDock Vina matches well with that of the cocrystallized ligand (root mean square deviation of the heavy atoms 1.45 Å). The locations and a general ligand binding interaction model for the LBS were determined, leading to the discovery of novel mitoNEET ligands. An in vitro fluorescence binding assay utilizing purified recombinant mitoNEET protein was used to determine the binding affinity of a predicted mitoNEET ligand, and the data obtained is in good agreement with AutoDock Vina results. The discovery of potential mitoNEET ligand binding sites and novel ligands, opens up the possibility for detailed structural studies of mitoNEET–ligand complexes, as well as rational design of novel ligands specifically targeted for mitoNEET. PMID:21531159

  10. Multifunctional Transmembrane Protein Ligands for Cell-Specific Targeting of Plasma Membrane-Derived Vesicles.

    PubMed

    Zhao, Chi; Busch, David J; Vershel, Connor P; Stachowiak, Jeanne C

    2016-07-01

    Liposomes and nanoparticles that bind selectively to cell-surface receptors can target specific populations of cells. However, chemical conjugation of ligands to these particles is difficult to control, frequently limiting ligand uniformity and complexity. In contrast, the surfaces of living cells are decorated with highly uniform populations of sophisticated transmembrane proteins. Toward harnessing cellular capabilities, here it is demonstrated that plasma membrane vesicles (PMVs) derived from donor cells can display engineered transmembrane protein ligands that precisely target cells on the basis of receptor expression. These multifunctional targeting proteins incorporate (i) a protein ligand, (ii) an intrinsically disordered protein spacer to make the ligand sterically accessible, and (iii) a fluorescent protein domain that enables quantification of the ligand density on the PMV surface. PMVs that display targeting proteins with affinity for the epidermal growth factor receptor (EGFR) bind at increasing concentrations to breast cancer cells that express increasing levels of EGFR. Further, as an example of the generality of this approach, PMVs expressing a single-domain antibody against green fluorescence protein (eGFP) bind to cells expressing eGFP-tagged receptors with a selectivity of ≈50:1. The results demonstrate the versatility of PMVs as cell targeting systems, suggesting diverse applications from drug delivery to tissue engineering. PMID:27294846

  11. Scintillation Proximity Radioimmunoassay Utilizing 125I-Labeled Ligands

    NASA Astrophysics Data System (ADS)

    Udenfriend, Sidney; Diekmann Gerber, Louise; Brink, Larry; Spector, Sydney

    1985-12-01

    A unique type of radioimmunoassay is described that does not require centrifugation or separation. Microbeads containing a fluorophor are covalently linked to antibody. When an 125I-labeled antigen is added it binds to the beads and, by its proximity, the emitted short-range electrons of the 125I excite the fluorophor in the beads. The light emitted can be measured in a standard scintillation counter. Addition of unlabeled antigen from tissue extracts displaces the labeled ligand and diminishes the fluorescent signal. Application of scintillation proximity immunoassay to tissue enkephalins, serum thyroxin, and urinary morphine is described. Applications of the principle to study the kinetics of interaction between receptors and ligands are discussed.

  12. Scintillation proximity radioimmunoassay utilizing 125I-labeled ligands

    SciTech Connect

    Udenfriend, S.; Gerber, L.D.; Brink, L.; Spector, S.

    1985-12-01

    A unique type of radioimmunoassay is described that does not require centrifugation or separation. Microbeads containing a fluorophor are covalently linked to antibody. When an /sup 125/I-labeled antigen is added it binds to the beads and, by its proximity, the emitted short-range electrons of the /sup 125/I excite the fluorophor in the beads. The light emitted can be measured in a standard scintillation counter. Addition of unlabeled antigen from tissue extracts displaces the labeled ligand and diminishes the fluorescent signal. Application of scintillation proximity immunoassay to tissue enkephalins, serum thyroxin, and urinary morphine is described. Applications of the principle to study the kinetics of interaction between receptors and ligands are discussed.

  13. A Fluorescence Lecture Demonstration.

    ERIC Educational Resources Information Center

    Bozzelli, Joseph W.; Kemp, Marwin

    1982-01-01

    Describes fluorescence demonstrations related to several aspects of molecular theory and quantitized energy levels. Demonstrations use fluorescent chemical solutions having luminescence properties spanning the visible spectrum. Also describes a demonstration of spontaneous combustion of familiar substances in chlorine. (JN)

  14. Fluorescent optical position sensor

    DOEpatents

    Weiss, Jonathan D.

    2005-11-15

    A fluorescent optical position sensor and method of operation. A small excitation source side-pumps a localized region of fluorescence at an unknown position along a fluorescent waveguide. As the fluorescent light travels down the waveguide, the intensity of fluorescent light decreases due to absorption. By measuring with one (or two) photodetectors the attenuated intensity of fluorescent light emitted from one (or both) ends of the waveguide, the position of the excitation source relative to the waveguide can be determined by comparing the measured light intensity to a calibrated response curve or mathematical model. Alternatively, excitation light can be pumped into an end of the waveguide, which generates an exponentially-decaying continuous source of fluorescent light along the length of the waveguide. The position of a photodetector oriented to view the side of the waveguide can be uniquely determined by measuring the intensity of the fluorescent light emitted radially at that location.

  15. Safe biodegradable fluorescent particles

    DOEpatents

    Martin, Sue I.; Fergenson, David P.; Srivastava, Abneesh; Bogan, Michael J.; Riot, Vincent J.; Frank, Matthias

    2010-08-24

    A human-safe fluorescence particle that can be used for fluorescence detection instruments or act as a safe simulant for mimicking the fluorescence properties of microorganisms. The particle comprises a non-biological carrier and natural fluorophores encapsulated in the non-biological carrier. By doping biodegradable-polymer drug delivery microspheres with natural or synthetic fluorophores, the desired fluorescence can be attained or biological organisms can be simulated without the associated risks and logistical difficulties of live microorganisms.

  16. Atmospheric Nitrogen Fluorescence Yield

    NASA Technical Reports Server (NTRS)

    Adams, J. H., Jr.; Christl, M. J.; Fountain, W. F.; Gregory, J. C.; Martens, K. U.; Sokolsky, Pierre; Whitaker, Ann F. (Technical Monitor)

    2001-01-01

    Several existing and planned experiments estimate the energies of ultra-high energy cosmic rays from air showers using the atmospheric nitrogen fluorescence. The nitrogen fluorescence yield from air shower electrons depends on the atmospheric composition. We will discuss the uncertainties in the fluorescence yield form electrons in the real atmosphere and describe a concept for a small balloon payload to measure the atmospheric fluorescence yield as a function of attitude.

  17. Oxidative Stress Promotes Ligand-independent and Enhanced Ligand-dependent Tumor Necrosis Factor Receptor Signaling*

    PubMed Central

    Ozsoy, Hatice Z.; Sivasubramanian, Natarajan; Wieder, Eric D.; Pedersen, Steen; Mann, Douglas L.

    2008-01-01

    Tumor necrosis factor (TNF) receptor 1 (TNFR1, p55) and 2 (TNFR2, p75) are characterized by several cysteine-rich modules in the extracellular domain, raising the possibility that redox-induced modifications of these cysteine residues might alter TNFR function. To test this possibility, we examined fluorescence resonance energy transfer (FRET) in 293T cells transfected with CFP- and YFP-tagged TNFRs exposed to the thiol oxidant diamide. Treatment with high concentrations of diamide (1 mm) resulted in an increase in the FRET signal that was sensitive to inhibition with the reducing agent dithiothreitol, suggesting that oxidative stress resulted in TNFR self-association. Treatment of cells with low concentrations of diamide (1 μm) that was not sufficient to provoke TNFR self-association resulted in increased TNF-induced FRET signals relative to the untreated cells, suggesting that oxidative stress enhanced ligand-dependent TNFR signaling. Similar findings were obtained when the TNFR1- and TNFR2-transfected cells were pretreated with a cell-impermeable oxidase, DsbA, that catalyzes disulfide bond formation between thiol groups on cysteine residues. The changes in TNFR self-association were functionally significant, because pretreating the HeLa cells and 293T cells resulted in increased TNF-induced NF-κB activation and TNF-induced expression of IκB and syndecan-4 mRNA levels. Although pretreatment with DsbA did not result in an increase in TNF binding to TNFRs, it resulted in increased TNF-induced activation of NF-κB, consistent with an allosteric modification of the TNFRs. Taken together, these results suggest that oxidative stress promotes TNFR receptor self-interaction and ligand-independent and enhanced ligand-dependent TNF signaling. PMID:18544535

  18. Origins of concentration dependence of waiting times for single-molecule fluorescence binding.

    PubMed

    Yang, Jin; Pearson, John E

    2012-06-28

    Binary fluorescence time series obtained from single-molecule imaging experiments can be used to infer protein binding kinetics, in particular, association and dissociation rate constants from waiting time statistics of fluorescence intensity changes. In many cases, rate constants inferred from fluorescence time series exhibit nonintuitive dependence on ligand concentration. Here, we examine several possible mechanistic and technical origins that may induce ligand dependence of rate constants. Using aggregated Markov models, we show under the condition of detailed balance that non-fluorescent bindings and missed events due to transient interactions, instead of conformation fluctuations, may underly the dependence of waiting times and thus apparent rate constants on ligand concentrations. In general, waiting times are rational functions of ligand concentration. The shape of concentration dependence is qualitatively affected by the number of binding sites in the single molecule and is quantitatively tuned by model parameters. We also show that ligand dependence can be caused by non-equilibrium conditions which result in violations of detailed balance and require an energy source. As to a different but significant mechanism, we examine the effect of ambient buffers that can substantially reduce the effective concentration of ligands that interact with the single molecules. To demonstrate the effects by these mechanisms, we applied our results to analyze the concentration dependence in a single-molecule experiment EGFR binding to fluorophore-labeled adaptor protein Grb2 by Morimatsu et al. [Proc. Natl. Acad. Sci. U.S.A. 104, 18013 (2007)]. PMID:22755586

  19. Influence of fluorescent tag on the motility properties of kinesin-1 in single-molecule assays.

    PubMed

    Norris, Stephen R; Núñez, Marcos F; Verhey, Kristen J

    2015-03-10

    Molecular motors such as kinesin and dynein use the energy derived from ATP hydrolysis to walk processively along microtubule tracks and transport various cargoes inside the cell. Recent advancements in fluorescent protein (FP) research enable motors to be fluorescently labeled such that single molecules can be visualized inside cells in multiple colors. The performance of these fluorescent tags can vary depending on their spectral properties and a natural tendency for oligomerization. Here we present a survey of different fluorescent tags fused to kinesin-1 and studied by single-molecule motility assays of mammalian cell lysates. We tested eight different FP tags and found that seven of them display sufficient fluorescence intensity and photostability to visualize motility events. Although none of the FP tags interfere with the enzymatic properties of the motor, four of the tags (EGFP, monomeric EGFP, tagRFPt, and mApple) cause aberrantly long motor run lengths. This behavior is unlikely to be due to electrostatic interactions and is probably caused by tag-dependent oligomerization events that appear to be facilitated by fusion to the dimeric kinesin-1. We also compared the single-molecule performance of various fluorescent SNAP and HALO ligands. We found that although both green and red SNAP ligands provide sufficient fluorescent signal, only the tetramethyl rhodamine (TMR) HALO ligand provides sufficient signal for detection in these assays. This study will serve as a valuable reference for choosing fluorescent labels for single-molecule motility assays. PMID:25762325

  20. RNA Fluorescence with Light-Up Aptamers

    PubMed Central

    Ouellet, Jonathan

    2016-01-01

    Seeing is not only believing; it also includes understanding. Cellular imaging with GFP in live cells has been transformative in many research fields. Modulation of cellular regulation is tightly regulated and innovative imaging technologies contribute to further understand cellular signaling and physiology. New types of genetically encoded biosensors have been developed over the last decade. They are RNA aptamers that bind with their cognate fluorogen ligands and activate their fluorescence. The emergence and the evolution of these RNA aptamers as well as their conversion into a wide spectrum of applications are examined in a global way. PMID:27446908

  1. RNA Fluorescence with Light-Up Aptamers.

    PubMed

    Ouellet, Jonathan

    2016-01-01

    Seeing is not only believing; it also includes understanding. Cellular imaging with GFP in live cells has been transformative in many research fields. Modulation of cellular regulation is tightly regulated and innovative imaging technologies contribute to further understand cellular signaling and physiology. New types of genetically encoded biosensors have been developed over the last decade. They are RNA aptamers that bind with their cognate fluorogen ligands and activate their fluorescence. The emergence and the evolution of these RNA aptamers as well as their conversion into a wide spectrum of applications are examined in a global way. PMID:27446908

  2. Fluorescence study of sugars

    NASA Astrophysics Data System (ADS)

    Thongjamroon, Sunida; Pattanaporkratana, Apichart

    2015-07-01

    We studied photoemission of monosaccharides and disaccharides using laser-induced fluorescence spectroscopy. A 532- nm, 10 mW, laser was used to excite the samples and back-scattering signals were collected by a spectrometer. We found that most sugars show weak fluorescence in solid phase but do not fluoresce when dissolved in water solutions. The emission spectra show similar peak intensity at 590 nm, but they are different in emission intensities. We suggest that the fluorescence spectra may be used to differentiate sugar type, even though the origin of the fluorescence is unclear and needed further study.

  3. Optical Response of Terpyridine Ligands to Zinc Binding: A Close Look at the Substitution Effect by Spectroscopic Studies at Low Temperature.

    PubMed

    Bi, Xiaoman; Pang, Yi

    2016-04-01

    Terpyridine (tpy) ligands are popular building blocks to bind metal ions. Several tpy ligands with different substituents were synthesized and examined for their binding with zinc cation. The study revealed a large substituent effect on the zinc binding-induced fluorescence quenching. With the aid of a liquid nitrogen Dewar, the tpy molecules were frozen to their ground-state conformation, preventing (or minimizing) molecular reorganization in the photoinduced excited state. This allowed us to detect the fluorescence spectra from the locally excited state (having a minimum of charge transfer interaction) and the temperature-dependent fluorescence. The fluorescence response to low temperature provided useful information about the intramolecular charge transfer (ICT) interaction between the donor and acceptor groups. Furthermore, a strong donor substituent (such as Me2N) played an essential role in observed fluorescence quenching. The study also provides a useful example to elucidate the ICT mechanism by using low-temperature fluorescence spectroscopy. PMID:26962883

  4. Fluorescent bionanoprobes to characterize cytoadhesion and cytoinvasion.

    PubMed

    Fillafer, Christian; Friedl, Daniela S; Wirth, Michael; Gabor, Franz

    2008-05-01

    To overcome current limitations in diagnostic imaging and targeted drug delivery, a highly versatile tool is presented that can be used to representatively investigate the effects of submicroparticles intended for the use in biological systems. An effective approach to render colloids trackable is developed by stable attachment of the fluorescent probe BODIPY 493/503 (BOD: 4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene) to a biodegradable and biocompatible particle core matrix. BOD submicroparticles are shown to be stable, can be surface modified, and exhibit high fluorescence emission. Upon conjugation with human serum albumin (nonspecific) and wheat germ agglutinin (biorecognitive) as model ligands explicit differences are found in the cytoadhesive and cytoinvasive characteristics of the submicroparticles using Caco-2 cells. These results demonstrate the potency of BOD-labeled colloids as a versatile analytical platform for a multifaceted investigation of cell-particle interactions in biological systems. PMID:18491364

  5. What are Nuclear Receptor Ligands?

    PubMed Central

    Sladek, Frances M.

    2010-01-01

    Nuclear receptors (NRs) are a family of highly conserved transcription factors that regulate transcription in response to small lipophilic compounds. They play a role in every aspect of development, physiology and disease in humans. They are also ubiquitous in and unique to the animal kingdom suggesting that they may have played an important role in their evolution. In contrast to the classical endocrine receptors that originally defined the family, recent studies suggest that the first NRs might have been sensors of their environment, binding ligands that were external to the host organism. The purpose of this review is to provide a broad perspective on NR ligands and address the issue of exactly what constitutes a NR ligand from historical, biological and evolutionary perspectives. This discussion will lay the foundation for subsequent reviews in this issue as well as pose new questions for future investigation. PMID:20615454

  6. Why mercury prefers soft ligands

    SciTech Connect

    Riccardi, Demian M; Guo, Hao-Bo; Gu, Baohua; Parks, Jerry M; Summers, Anne; Miller, S; Liang, Liyuan; Smith, Jeremy C

    2013-01-01

    Mercury (Hg) is a major global pollutant arising from both natural and anthropogenic sources. Defining the factors that determine the relative affinities of different ligands for the mercuric ion, Hg2+, is critical to understanding its speciation, transformation, and bioaccumulation in the environment. Here, we use quantum chemistry to dissect the relative binding free energies for a series of inorganic anion complexes of Hg2+. Comparison of Hg2+ ligand interactions in the gaseous and aqueous phases shows that differences in interactions with a few, local water molecules led to a clear periodic trend within the chalcogenide and halide groups and resulted in the well-known experimentally observed preference of Hg2+ for soft ligands such as thiols. Our approach establishes a basis for understanding Hg speciation in the biosphere.

  7. Molecular Recognition and Ligand Association

    NASA Astrophysics Data System (ADS)

    Baron, Riccardo; McCammon, J. Andrew

    2013-04-01

    We review recent developments in our understanding of molecular recognition and ligand association, focusing on two major viewpoints: (a) studies that highlight new physical insight into the molecular recognition process and the driving forces determining thermodynamic signatures of binding and (b) recent methodological advances in applications to protein-ligand binding. In particular, we highlight the challenges posed by compensating enthalpic and entropic terms, competing solute and solvent contributions, and the relevance of complex configurational ensembles comprising multiple protein, ligand, and solvent intermediate states. As more complete physics is taken into account, computational approaches increase their ability to complement experimental measurements, by providing a microscopic, dynamic view of ensemble-averaged experimental observables. Physics-based approaches are increasingly expanding their power in pharmacology applications.

  8. Excitation energy transfer in europium chelate with doxycycline in the presence of a second ligand in micellar solutions of nonionic surfactants

    NASA Astrophysics Data System (ADS)

    Smirnova, T. D.; Shtykov, S. N.; Kochubei, V. I.; Khryachkova, E. S.

    2011-01-01

    The complexation of Eu3+ with doxycycline (DC) antibiotic in the presence of several second ligands and surfactant micelles of different types is studied by the spectrophotometric and luminescence methods. It is found that the efficiency of excitation energy transfer in Eu3+-DC chelate depends on the nature of the second ligand and surfactant micelles. Using thenoyltrifluoroacetone (TTA) as an example, it is shown that the second ligand additionally sensitizes the europium fluorescence, and the possibility of intermediate sensitization of DC and then of europium is shown by the example of 1,10-phenanthroline. In all cases, the excitation energy transfer efficiency was increased due to the so-called antenna effect. The decay kinetics of the sensitized fluorescence of the binary and mixed-ligand chelates in aqueous and micellar solutions of nonionic surfactants is studied and the relative quantum yields and lifetimes of fluorescence are determined.

  9. Targeting Protein-Protein Interactions with Trimeric Ligands: High Affinity Inhibitors of the MAGUK Protein Family

    PubMed Central

    Nissen, Klaus B.; Haugaard-Kedström, Linda M.; Wilbek, Theis S.; Nielsen, Line S.; Åberg, Emma; Kristensen, Anders S.; Bach, Anders; Jemth, Per; Strømgaard, Kristian

    2015-01-01

    PDZ domains in general, and those of PSD-95 in particular, are emerging as promising drug targets for diseases such as ischemic stroke. We have previously shown that dimeric ligands that simultaneously target PDZ1 and PDZ2 of PSD-95 are highly potent inhibitors of PSD-95. However, PSD-95 and the related MAGUK proteins contain three consecutive PDZ domains, hence we envisioned that targeting all three PDZ domains simultaneously would lead to more potent and potentially more specific interactions with the MAGUK proteins. Here we describe the design, synthesis and characterization of a series of trimeric ligands targeting all three PDZ domains of PSD-95 and the related MAGUK proteins, PSD-93, SAP-97 and SAP-102. Using our dimeric ligands targeting the PDZ1-2 tandem as starting point, we designed novel trimeric ligands by introducing a PDZ3-binding peptide moiety via a cysteine-derivatized NPEG linker. The trimeric ligands generally displayed increased affinities compared to the dimeric ligands in fluorescence polarization binding experiments and optimized trimeric ligands showed low nanomolar inhibition towards the four MAGUK proteins, thus being the most potent inhibitors described. Kinetic experiments using stopped-flow spectrometry showed that the increase in affinity is caused by a decrease in the dissociation rate of the trimeric ligand as compared to the dimeric ligands, likely reflecting the lower probability of simultaneous dissociation of all three PDZ ligands. Thus, we have provided novel inhibitors of the MAGUK proteins with exceptionally high affinity, which can be used to further elucidate the therapeutic potential of these proteins. PMID:25658767

  10. Targeted delivery to bone and mineral deposits using bisphosphonate ligands.

    PubMed

    Cole, Lisa E; Vargo-Gogola, Tracy; Roeder, Ryan K

    2016-04-01

    The high concentration of mineral present in bone and pathological calcifications is unique compared with all other tissues and thus provides opportunity for targeted delivery of pharmaceutical drugs, including radiosensitizers and imaging probes. Targeted delivery enables accumulation of a high local dose of a therapeutic or imaging contrast agent to diseased bone or pathological calcifications. Bisphosphonates (BPs) are the most widely utilized bone-targeting ligand due to exhibiting high binding affinity to hydroxyapatite mineral. BPs can be conjugated to an agent that would otherwise have little or no affinity for the sites of interest. This article summarizes the current state of knowledge and practice for the use of BPs as ligands for targeted delivery to bone and mineral deposits. The clinical history of BPs is briefly summarized to emphasize the success of these molecules as therapeutics for metabolic bone diseases. Mechanisms of binding and the relative binding affinity of various BPs to bone mineral are introduced, including common methods for measuring binding affinity in vitro and in vivo. Current research is highlighted for the use of BP ligands for targeted delivery of BP conjugates in various applications, including (1) therapeutic drug delivery for metabolic bone diseases, bone cancer, other bone diseases, and engineered drug delivery platforms; (2) imaging probes for scintigraphy, fluorescence, positron emission tomography, magnetic resonance imaging, and computed tomography; and (3) radiotherapy. Last, and perhaps most importantly, key structure-function relationships are considered for the design of drugs with BP ligands, including the tether length between the BP and drug, the size of the drug, the number of BP ligands per drug, cleavable tethers between the BP and drug, and conjugation schemes. PMID:26482186

  11. Multifunctional Ligands in Transition Metal Catalysis

    SciTech Connect

    Crabtree, Robert H

    2011-01-01

    Sophisticated ligands are now being designed that do far more than just fulfil their traditional spectator roles by binding to the metal and providing a sterically-defined binding pocket for the substrate in homogeneous transition metal catalysis. This Focus review emphasizes selected cases in which ligands carry additional functional groups that change the properties of the ligand as a result of an external stimulus or undergo catalytically-relevant ligand-based reactivity. These include proton responsive ligands capable of gaining or losing one or more protons, ligands having a hydrogen bonding function, electroresponsive ligands capable of gaining or losing one or more electrons, and photoresponsive ligands capable of undergoing a useful change of properties upon irradiation. Molecular recognition ligands and proton coupled electron transfer (PCET) are briefly discussed.

  12. Fluorescent magnetic hybrid nanoprobe for multimodal bioimaging

    NASA Astrophysics Data System (ADS)

    Koktysh, Dmitry; Bright, Vanessa; Pham, Wellington

    2011-07-01

    A fluorescent magnetic hybrid imaging nanoprobe (HINP) was fabricated by the conjugation of superparamagnetic Fe3O4 nanoparticles and visible light emitting (~600 nm) fluorescent CdTe/CdS quantum dots (QDs). The assembly strategy used the covalent linking of the oxidized dextran shell of magnetic particles to the glutathione ligands of QDs. The synthesized HINP formed stable water-soluble colloidal dispersions. The structure and properties of the particles were characterized by transmission electron and atomic force microscopy, energy dispersive x-ray analysis and inductively coupled plasma optical emission spectroscopy, dynamic light scattering analysis, optical absorption and photoluminescence spectroscopy, and fluorescent imaging. The luminescence imaging region of the nanoprobe was extended to the near-infrared (NIR) (~800 nm) by conjugation of the superparamagnetic nanoparticles with synthesized CdHgTe/CdS QDs. Cadmium, mercury based QDs in HINP can be easily replaced by novel water-soluble glutathione stabilized AgInS2/ZnS QDs to present a new class of cadmium-free multimodal imaging agents. The observed NIR photoluminescence of fluorescent magnetic nanocomposites supports their use for bioimaging. The developed HINP provides dual-imaging channels for simultaneous optical and magnetic resonance imaging.

  13. Fluorescent indicator dyes for calcium ions

    NASA Technical Reports Server (NTRS)

    Tsien, Roger Y. (Inventor); Grynkiewicz, Grzegorz (Inventor)

    1986-01-01

    The present invention discloses a new class of highly fluorescent indicator dyes that are specific for calcium ions. The new fluorescent indicator dyes combine a stilbene-type fluorophore with a tetracarboxylate parent Ca.sup.2+ chelating compound having the octacoordinate pattern of liganding groups characteristic of EGTA and BAPTA. Preferred forms contain extra heterocyclic bridges to reinforce the ethylenic bond of the stilbene and to reduce hydrophobicity. Compared to their widely used predecessor, quin2, the new dyes offer up to thirty-fold brighter fluorescence, major changes in wavelength (not just intensity) upon Ca.sup.2+ binding, slightly lower affinities for Ca.sup.2+, slightly longer wavelengths of excitation, and considerably improved selectivity for Ca.sup.2+ over other divalent cations. These properties, particularly the wavelength sensitivity to Ca.sup.2+, make the dyes useful indicators for many intracellular applications, especially in single cells, adherent cell layers, or bulk tissues. The present invention also discloses an improved method for synthesizing alpha-acyloxyalkyl bromides wherein the bromides so synthesized are free of contaminating bis(1-bromoalkyl)ether. The improved method is exemplified herein in the synthesis of acetoxymethyl bromide, a compound useful in preparing the acetoxymethyl esters disclosed herein as novel Ca.sup.2+ specific fluorescent indicators.

  14. Fluorescent magnetic hybrid nanoprobe for multimodal bioimaging

    PubMed Central

    Bright, Vanessa

    2011-01-01

    A fluorescent magnetic hybrid imaging nanoprobe (HINP) was fabricated by conjugation of superparamagnetic Fe3O4 nanoparticles and visible light-emitting (~600 nm) fluorescent CdTe/CdS quantum dots (QDs). The assembly strategy used the covalent linking of the oxidized dextran shell of magnetic particles to the glutathione ligands of QDs. Synthesized HINP formed stable water-soluble colloidal dispersions. The structure and properties of the particles were characterized by transmission electron and atomic force microscopy, energy dispersive X-ray analysis and inductively coupled plasma optical emission spectroscopy, dynamic light scattering analysis, optical absorption and photoluminescence spectroscopy, and fluorescent imaging. The luminescence imaging region of the nanoprobe was extended to the near-infrared (NIR) (~800 nm) by conjugation of superparamagnetic nanoparticles with synthesized CdHgTe/CdS QDs. Cadmium, mercury based QDs in HINP can be easily replaced by novel water soluble glutathione stabilized AgInS2/ZnS QDs to present a new class of cadmium-free multimodal imaging agents. Observed NIR photoluminescence of fluorescent magnetic nanocomposites supports their use for bioimaging. The developed HINP provides dual-imaging channels for simultaneous optical and magnetic resonance imaging. PMID:21597146

  15. Phosphate-induced fluorescence of a tetraphenylethene-substituted tripodal tris(urea) receptor.

    PubMed

    Zhao, Jie; Yang, Dong; Zhao, Yanxia; Cao, Liping; Zhang, Zhibin; Yang, Xiao-Juan; Wu, Biao

    2016-04-25

    A tetraphenylethene (TPE)-decorated tripodal tris(urea) ligand was synthesized, which shows large emission enhancement when binding to an orthophosphate anion (PO4(3-)), but exhibits only weak or no fluorescence with other anions. The anion-binding and fluorescence properties were studied by X-ray crystal structure, NMR and fluorescence spectroscopy, and by DFT computations and the results demonstrate that the different fluorescence performance may be determined by the anion-binding modes (i.e., full- or half-encapsulation). PMID:27028977

  16. Tropolone Complexes Formed with Amphoteric Ligands: Structure and Dynamics as Viewed across the Vibronic Landscape

    NASA Astrophysics Data System (ADS)

    Nemchick, Deacon J.; Chew, Kathryn; Vaccaro, Patrick H.

    2013-06-01

    Owing to the presence of a finite potential barrier that adjoins hydroxylic (proton-donating) and ketonic (proton-accepting) oxygen atom centers, tropolone (TrOH) long has served as a model system for the investigation of coherent (symmetrical) proton-transfer events. Hydrogen-bound complexes formed by docking amphoteric species onto the TrOH substrate, such as those involving formic acid [TrOH-(FA)_n] and other simultaneous donor-acceptor ligands, have been generated under supersonic free-jet expansion conditions. For binary adducts (n=1), quantum-chemical calculations predict two nearly degenerate isomers that can be labeled as external (ligand attached to the seven-membered aromatic ring) and internal (ligand bound to the O-H \\cdot\\cdot\\cdot O reaction site), where the latter cleft-bound form offers the tantalizing possibility of undergoing a double proton-transfer process. A variety of spectroscopic probes build around the intense ˜{A}^{1}{B}_{2}-˜{X}^{1}{A}_{1} (π ^{*}←π) near-ultraviolet absorption feature of bare tropolone have been enlisted to elucidate the binding motifs and reaction pathways in complexes containing one or more amphoteric ligands, including vibrationally resolved schemes based upon laser-induced fluorescence (LIF), dispersed fluorescence (DF), and fluorescence hole-burning (FHB) methods. Structural and dynamical information gleaned from these experiments will be discussed in light of complementary ab initio calculations.

  17. "Ligands-with-Benefits": Naphthalene-Substituted Schiff Bases Yielding New Ni(II) Metal Clusters with Ferromagnetic and Emissive Properties and Undergoing Exciting Transformations.

    PubMed

    Perlepe, Panagiota S; Cunha-Silva, Luís; Gagnon, Kevin J; Teat, Simon J; Lampropoulos, Christos; Escuer, Albert; Stamatatos, Theocharis C

    2016-02-01

    The initial employment of the fluorescent bridging ligand N-naphthalidene-2-amino-5-chlorobenzoic acid (nacbH2) in metal cluster chemistry has led to new Ni12 (1) and Ni5 (2) clusters with wheel-like and molecular-chain topologies, respectively. The doubly-deprotonated nacb(2-) ligands were found to adopt four different coordination modes within 1 and 2. The nature of the ligand has also allowed unexpected organic transformations to occur and ferromagnetic and emission behaviors to emerge. The combined work demonstrates the ability of some "ligands-with-benefits" to yield beautiful structures with exciting topologies and interesting physicochemical properties. PMID:26788587

  18. Synthesis and Fluorescence Properties of Eu(3+), Tb(3+) Complexes with Schiff Base Derivatives.

    PubMed

    Liu, Yanhong; Kong, Weihua; Yang, Zehui; Dai, Ming; Shi, Ling; Guo, Dongcai

    2016-03-01

    Novel Schiff base ligands derived from N'-benzylidene-benzohydrazide (substituted by -H, -CH3, -OCH3, -Cl) and 2-chloro-N-phenylacetamide were synthesized. The solid complexes of rare earth (Eu, Tb) nitrate with these Schiff base ligands were synthesized and characterized by elemental analysis, EDTA titrimetric analysis, thermal analysis, infrared spectra and UV-Vis spectra analysis. The fluorescence properties of rare earth (Eu, Tb) complexes in solid were studied. Under the excitation of ultraviolet light, these complexes exhibited characteristic emission of europium and terbium ions. The results showed that the ligand favored energy transfer to the emitting energy of Eu and Tb ions. Effects of different ligands on the fluorescence intensity of rare earth (Eu, Tb) complexes had been discussed. The electrochemical properties of rare earth (Eu, Tb) complexes were also investigated. PMID:26658796

  19. Polypharmacology of dopamine receptor ligands.

    PubMed

    Butini, S; Nikolic, K; Kassel, S; Brückmann, H; Filipic, S; Agbaba, D; Gemma, S; Brogi, S; Brindisi, M; Campiani, G; Stark, H

    2016-07-01

    Most neurological diseases have a multifactorial nature and the number of molecular mechanisms discovered as underpinning these diseases is continuously evolving. The old concept of developing selective agents for a single target does not fit with the medical need of most neurological diseases. The development of designed multiple ligands holds great promises and appears as the next step in drug development for the treatment of these multifactorial diseases. Dopamine and its five receptor subtypes are intimately involved in numerous neurological disorders. Dopamine receptor ligands display a high degree of cross interactions with many other targets including G-protein coupled receptors, transporters, enzymes and ion channels. For brain disorders like Parkinsońs disease, schizophrenia and depression the dopaminergic system, being intertwined with many other signaling systems, plays a key role in pathogenesis and therapy. The concept of designed multiple ligands and polypharmacology, which perfectly meets the therapeutic needs for these brain disorders, is herein discussed as a general ligand-based concept while focusing on dopaminergic agents and receptor subtypes in particular. PMID:27234980

  20. Fluorescence biosensing strategy based on energy transfer between fluorescently labeled receptors and a metallic surface.

    PubMed

    Pérez-Luna, Víctor H; Yang, Saipeng; Rabinovich, Emmanuil M; Buranda, Tione; Sklar, Larry A; Hampton, Philip D; López, Gabriel P

    2002-01-01

    A new fluorescence-based biosensor is presented. The biosensing scheme is based on the fact that a fluorophore in close proximity to a metal film (<100 A) experiences strong quenching of fluorescence and a dramatic reduction in the lifetime of the excited state. By immobilizing the analyte of interest (or a structural analog of the analyte) to a metal surface and exposing it to a labeled receptor (e.g. antibody), the fluorescence of the labeled receptor becomes quenched upon binding because of the close proximity to the metal. Upon exposure to free analyte, the labeled receptor dissociates from the surface and diffuses into the bulk of the solution. This increases its separation from the metal and an increase of fluorescence intensity and/or lifetime of the excited state is observed that indicates the presence of the soluble analyte. By enclosing this system within a small volume with a semipermeable membrane, a reversible device is obtained. We demonstrate this scheme using a biotinylated self-assembled monolayer (SAM) on gold as our surface immobilized analyte analog, fluorescently labeled anti-biotin as a receptor, and a solution of biotin in PBS as a model analyte. This scheme could easily be extended to transduce a wide variety of protein-ligand interactions and other biorecognition phenomena (e.g. DNA hybridization) that result in changes in the architecture of surface immobilized biomolecules such that a change in the separation distance between fluorophores and the metal film is obtained. PMID:11742737

  1. Evaluation of fluorescence-based thermal shift assays for hit identification in drug discovery.

    PubMed

    Lo, Mei-Chu; Aulabaugh, Ann; Jin, Guixian; Cowling, Rebecca; Bard, Jonathan; Malamas, Michael; Ellestad, George

    2004-09-01

    The fluorescence-based thermal shift assay is a general method for identification of inhibitors of target proteins from compound libraries. Using an environmentally sensitive fluorescent dye to monitor protein thermal unfolding, the ligand-binding affinity can be assessed from the shift of the unfolding temperature (Delta Tm) obtained in the presence of ligands relative to that obtained in the absence of ligands. In this article, we report that the thermal shift assay can be conducted in an inexpensive, commercially available device for temperature control and fluorescence detection. The binding affinities obtained from thermal shift assays are compared with the binding affinities measured by isothermal titration calorimetry and with the IC(50) values from enzymatic assays. The potential pitfalls in the data analysis of thermal shift assays are also discussed. PMID:15301960

  2. Binding phenomena and fluorescence quenching. II: Photophysics of aromatic residues and dependence of fluorescence spectra on protein conformation

    NASA Astrophysics Data System (ADS)

    Callis, Patrik R.

    2014-12-01

    The three amino acids with aromatic ring side chains-phenylalanine (Phe), tyrosine (Tyr), and especially tryptophan (Trp) have played a long and productive role in helping unlock the secrets of protein behavior by optical spectroscopy (absorption, fluorescence, circular dichroism, etc.) In principle, an appropriately placed Trp will undergo fluorescence wavelength and/or intensity changes upon whatever functional process a protein performs. Although perceived to be enigmatic and not well understood, Trp is arguably now better understood than many of the extrinsic probes currently in use. Basic principles of intrinsic tryptophan fluorescence quenching and wavelength shifts in proteins are presented, with strong emphasis on the importance of electrostatics. The condensed description of findings from recent experiments and simulations of tryptophan fluorescence and intrinsic quenching in proteins is designed to help authors in planning and interpreting experimental results of ligand binding studies.

  3. Nanomaterials can Dynamically Steer Cell Responses to Biological Ligands

    PubMed Central

    Sharma, Ram I.; Schwarzbauer, Jean E.; Moghe, Prabhas V.

    2011-01-01

    Traditional tissue regeneration approaches to activate cell behaviours on biomaterials rely on the use of extracellular matrix based or soluble growth factor cues. In this article, we highlight a novel approach to dynamically steer cellular phenomena such as cell motility based on nanoscale substratum features of biological ligands. Albumin derived nanocarriers (ANCs) of variable nanoscale size features were functionalized with fibronectin III9–10 matrix ligand and effects on primary human keratinocyte activation were investigated. The display of fibronectin fragment from ANCs significantly enhanced cell migration compared to free ligands at equivalent concentrations. Notably, cell migration was influenced by the size of underlying ANCs even for variably sized ANCs presenting comparable levels of fibronectin fragment. For equivalent ligand concentrations, cell migration on the smaller-sized ANCs (30 nm and 50 nm) was significantly more enhanced compared to that on larger-sized ANCs (75 nm and 100 nm). In contrast, the enhancement of cell migration on nanocarriers was abolished by the use of immobilized biofunctionalized ANCs, indicating that “dynamic” nanocarrier internalization events underlie the role of nanocarrier geometry on the differential regulation of cell migration kinetics. Uptake studies using fluorescent ANCs indicated that larger-sized ANCs showed delayed endocytic kinetics and hence could present barriers for internalization during the cell adhesion and motility processes. Motile cells exhibited diminished migration upon exposure to clathrin-inhibitors, but not caveolin-inhibitors, suggesting the role of clathrin-mediated endocytosis in facilitating cell migratory responsiveness to the nanocarriers. Overall, a monotonic relationship was found between the degree of nanocarrier cytointernalization rate and cell migration rate, suggesting the possibility of designing biointerfacial features for dynamic control of cell migration. Thus, the major

  4. A Bio-inspired Approach for Chromophore Communication: Ligand-to-Ligand and Host-to-Guest Energy Transfer in Hybrid Crystalline Scaffolds.

    PubMed

    Dolgopolova, Ekaterina A; Williams, Derek E; Greytak, Andrew B; Rice, Allison M; Smith, Mark D; Krause, Jeanette A; Shustova, Natalia B

    2015-11-01

    Efficient multiple-chromophore coupling in a crystalline metal-organic scaffold was achieved by mimicking a protein system possessing 100% energy-transfer (ET) efficiency between a green fluorescent protein variant and cytochrome b562. The two approaches developed for ET relied on the construction of coordination assemblies and host-guest coupling. Based on time-resolved photoluminescence measurements in combination with calculations of the spectral overlap function and Förster radius, we demonstrated that both approaches resulted in a very high ET efficiency. In particular, the observed ligand-to-ligand ET efficiency value was the highest reported so far for two distinct ligands in a metal-organic framework. These studies provide important insights for the rational design of crystalline hybrid scaffolds consisting of a large ensemble of chromophore molecules with the capability of directional ET. PMID:26377245

  5. Fluorescence Live Cell Imaging

    PubMed Central

    Ettinger, Andreas

    2014-01-01

    Fluorescence microscopy of live cells has become an integral part of modern cell biology. Fluorescent protein tags, live cell dyes, and other methods to fluorescently label proteins of interest provide a range of tools to investigate virtually any cellular process under the microscope. The two main experimental challenges in collecting meaningful live cell microscopy data are to minimize photodamage while retaining a useful signal-to-noise ratio, and to provide a suitable environment for cells or tissues to replicate physiological cell dynamics. This chapter aims to give a general overview on microscope design choices critical for fluorescence live cell imaging that apply to most fluorescence microscopy modalities, and on environmental control with a focus on mammalian tissue culture cells. In addition, we provide guidance on how to design and evaluate fluorescent protein constructs by spinning disk confocal microscopy. PMID:24974023

  6. Fluorescent fiber diagnostics

    DOEpatents

    Toeppen, John S.

    1994-10-04

    A fluorescent fiber (13) having a doped core (16) is pumped (11) by light (18) of a relatively short wavelength to produce fluorescence at a longer wavelength that is detected by detector (24). The level of fluorescence is monitored (26) and evaluated to provide information as to the excitation of the fiber (13) or the environment thereof. In particular, the level of intensity of the detected fluorescence may be used to measure the intensity of a light beam (18) passing axially through an optical fiber system (12) (FIG. 1 ), or the intensity of a light beam (46) passing radially through a fluorescent fiber (13) (FIG. 2 ), or the level of a fluid (32) in a tank (31) (FIG. 3 ), or a scintillation event (37) in a fluorescent fiber (13) pumped to produce amplification of the scintillation event (FIG. 4 ).

  7. Fluorescent fiber diagnostics

    DOEpatents

    Toeppen, John S.

    1994-01-01

    A fluorescent fiber (13) having a doped core (16) is pumped (11) by light (18) of a relatively short wavelength to produce fluorescence at a longer wavelength that is detected by detector (24). The level of fluorescence is monitored (26) and evaluated to provide information as to the excitation of the fiber (13) or the environment thereof. In particular, the level of intensity of the detected fluorescence may be used to measure the intensity of a light beam (18) passing axially through an optical fiber system (12) (FIG. 1 ), or the intensity of a light beam (46) passing radially through a fluorescent fiber (13) (FIG. 2 ), or the level of a fluid (32) in a tank (31) (FIG. 3 ), or a scintillation event (37) in a fluorescent fiber (13) pumped to produce amplification of the scintillation event (FIG. 4 ).

  8. A combinatorial biophysical approach; FTSA and SPR for identifying small molecule ligands and PAINs.

    PubMed

    Redhead, M; Satchell, R; Morkūnaitė, V; Swift, D; Petrauskas, V; Golding, E; Onions, S; Matulis, D; Unitt, J

    2015-06-15

    Biophysical methods have emerged as attractive screening techniques in drug discovery both as primary hit finding methodologies, as in the case of weakly active compounds such as fragments, and as orthogonal methods for hit validation for compounds discovered through conventional biochemical or cellular assays. Here we describe a dual method employing fluorescent thermal shift assay (FTSA), also known as differential scanning fluorimetry (DSF) and surface plasmon resonance (SPR), to interrogate ligands of the kinase p38α as well as several known pan-assay interference compounds (PAINs) such as aggregators, redox cyclers, and fluorescence quenchers. This combinatorial approach allows for independent verification of several biophysical parameters such as KD, kon, koff, ΔG, ΔS, and ΔH, which may further guide chemical development of a ligand series. Affinity values obtained from FTSA curves allow for insight into compound binding compared with reporting shifts in melting temperature. Ligand-p38 interaction data were in good agreement with previous literature. Aggregators and fluorescence quenchers appeared to reduce fluorescence signal in the FTSAs, causing artificially high shifts in Tm values, whereas redox compounds caused either shifts in affinity that did not agree between FTSA and SPR or a depression of FTSA signal. PMID:25837771

  9. Botany: floral fluorescence effect.

    PubMed

    Gandía-Herrero, Fernando; García-Carmona, Francisco; Escribano, Josefa

    2005-09-15

    The way flowers appear to insects is crucial for pollination. Here we describe an internal light-filtering effect in the flowers of Mirabilis jalapa, in which the visible fluorescence emitted by one pigment, a yellow betaxanthin, is absorbed by another, a violet betacyanin, to create a contrasting fluorescent pattern on the flower's petals. This finding opens up new possibilities for pollinator perception as fluorescence has not previously been considered as a potential signal in flowers. PMID:16163341

  10. Fluorescent minerals, a review

    USGS Publications Warehouse

    Modreski, P.J.; Aumente-Modreski, R.

    1996-01-01

    Fluorescent minerals are more than just an attractive novelty, and collecting them is a speciality for thousands of individuals who appreciate their beauty, rarity, and scientific value. Fluorescent properties can be used as an aid to mineral identification, locality determination, and distinction between natural and synthetic gemstones. This article gives an overview of those aspects of fluorescence that are of most interest to collectors, hobbyists, and mineralogists. -from Authors

  11. Fluorescent Methods to Study Transcription Initiation and Transition into Elongation

    PubMed Central

    Deshpande, Aishwarya P.; Sultana, Shemaila

    2015-01-01

    The DNA-dependent RNA polymerases induce specific conformational changes in the promoter DNA during transcription initiation. Fluorescence spectroscopy sensitively monitors these DNA conformational changes in real time and at equilibrium providing powerful ways to estimate interactions in transcriptional complexes and to assess how transcription is regulated by the promoter DNA sequence, transcription factors, and small ligands. Ensemble fluorescence methods described here probe the individual steps of promoter binding, bending, opening, and transition into the elongation using T7 phage and mitochondrial transcriptional systems as examples. PMID:25095993

  12. Overcoming compound fluorescence in the FLiK screening assay with red-shifted fluorophores.

    PubMed

    Schneider, Ralf; Gohla, Anne; Simard, Jeffrey R; Yadav, Dharmendra B; Fang, Zhizhou; van Otterlo, Willem A L; Rauh, Daniel

    2013-06-01

    In the attempt to discover novel chemical scaffolds that can modulate the activity of disease-associated enzymes, such as kinases, biochemical assays are usually deployed in high-throughput screenings. First-line assays, such as activity-based assays, often rely on fluorescent molecules by measuring a change in the total emission intensity, polarization state, or energy transfer to another fluorescent molecule. However, under certain conditions, intrinsic compound fluorescence can lead to difficult data analysis and to false-positive, as well as false-negative, hits. We have reported previously on a powerful direct binding assay called fluorescent labels in kinases ('FLiK'), which enables a sensitive measurement of conformational changes in kinases upon ligand binding. In this assay system, changes in the emission spectrum of the fluorophore acrylodan, induced by the binding of a ligand, are translated into a robust assay readout. However, under the excitation conditions of acrylodan, intrinsic compound fluorescence derived from highly conjugated compounds complicates data analysis. We therefore optimized this method by identifying novel fluorophores that excite in the far red, thereby avoiding compound fluorescence. With this advancement, even rigid compounds with multiple π-conjugated ring systems can now be measured reliably. This study was performed on three different kinase constructs with three different labeling sites, each undergoing distinct conformational changes upon ligand binding. It may therefore serve as a guideline for the establishment of novel fluorescence-based detection assays. PMID:23672540

  13. Fluorescence lifetime imaging of coral fluorescent proteins.

    PubMed

    Cox, Guy; Matz, Mikhail; Salih, Anya

    2007-03-01

    Corals, like many other coelenterates, contain fluorescent pigments that show considerable homology with the well known green fluorescent protein of the jellyfish Aequoria. In corals, unlike jellyfish, multiple proteins are present and the range of excitations and emissions suggest the possibility of energy transfer. The occurrence of Förster resonant energy transfer (FRET) between fluorescent proteins in corals has already been reported and time-resolved spectra have shown the effect on fluorescent lifetime, but without any spatial resolution. Lifetime confocal microscopy offers lower time resolution but excellent spatial resolution. Lifetimes of the isolated A. millepora pigments amilFP490, amilFP504, and amilFP593 (names indicate emission peaks) were 2.8, 2.9, and 2.9 ns, respectively. In the coral sample, imaging the entire emission spectrum from 420 nm, the mean lifetime was reduced to 1.5 ns, implying that FRET was occurring. Looking just at the fluorescence from FRET donors the lifetime was even shorter, at 1.3 ns, supporting this interpretation. In contrast, no reduction in lifetime is seen in the coral Euphyllia ancora, where the pigment distribution also suggests that the pigments are unlikely to be involved in photoprotection. This study set out to determine the extent of FRET between pigments in two corals, Acropora millepora and Euphyllia, ancora which differ in the arrangement of their pigments and hence possibly in pigment function. PMID:17279514

  14. Biphasic binding kinetics between FepA and its ligands.

    PubMed

    Payne, M A; Igo, J D; Cao, Z; Foster, S B; Newton, S M; Klebba, P E

    1997-08-29

    The Escherichia coli FepA protein is an energy- and TonB-dependent, ligand-binding porin that functions as a receptor for the siderophore ferric enterobactin and colicins B and D. We characterized the kinetic and thermodynamic parameters associated with the initial, energy-independent steps in ligand binding to FepA. In vivo experiments produced Kd values of 24, 185, and 560 nM for ferric enterobactin, colicin B, and colicin D, respectively. The siderophore and colicin B bound to FepA with a 1:1 stoichiometry, but colicin D bound to a maximum level that was 3-fold lower. Preincubation with ferric enterobactin prevented colicin B binding, and preincubation with colicin B prevented ferric enterobactin binding. Colicin B release from FepA was unexpectedly slow in vivo, about 10-fold slower than ferric enterobactin release. This slow dissociation of the colicin B.FepA complex facilitated the affinity purification of FepA and FepA mutants with colicin B-Sepharose. Analysis of a fluorescent FepA derivative showed that ferric enterobactin and colicin B adsorbed with biphasic kinetics, suggesting that both ligands bind in at least two distinct steps, an initial rapid stage and a subsequent slower step, that presumably establishes a transport-competent complex. PMID:9268330

  15. Using inositol as a biocompatible ligand for efficient transgene expression.

    PubMed

    Zhang, Lei; Bellis, Susan L; Fan, Yiwen; Wu, Yunkun

    2015-01-01

    Transgene transfection techniques using cationic polymers such as polyethylenimines (PEIs) and PEI derivatives as gene vectors have shown efficacy, although they also have shortcomings. PEIs have decent DNA-binding capability and good cell internalization performance, but they cannot deliver gene payloads very efficiently to cell nuclei. In this study, three hyperbranched polyglycerol-polyethylenimine (PG6-PEI) polymers conjugated with myo-inositol (INO) molecules were developed. The three resulting PG6-PEI-INO polymers have an increased number of INO ligands per molecule. PG6-PEI-INO 1 had only 14 carboxymethyl INO (CMINO) units per molecule. PG6-PEI-INO 2 had approximately 130 CMINO units per molecule. PG6-PEI-INO 3 had as high as 415 CMINO units approximately. Mixing PG6-PEI-INO polymers with DNA produced compact nanocomposites. We then performed localization studies using fluorescent microscopy. As the number of conjugated inositol ligands increased in PG6-PEI-INO polymers, there was a corresponding increase in accumulation of the polymers within 293T cell nuclei. Transfection performed with spherical 293T cells yielded 82% of EGFP-positive cells when using PG6-PEI-INO 3 as the vehicle. Studies further revealed that extracellular adenosine triphosphate (eATP) can inhibit the transgene efficiency of PG6-PEI-INO polymers, as compared with PEI and PG6-PEI that were not conjugated with inositol. Our work unveiled the possibility of using inositol as an effective ligand for transgene expression. PMID:25926732

  16. Using inositol as a biocompatible ligand for efficient transgene expression

    PubMed Central

    Zhang, Lei; Bellis, Susan L; Fan, Yiwen; Wu, Yunkun

    2015-01-01

    Transgene transfection techniques using cationic polymers such as polyethylenimines (PEIs) and PEI derivatives as gene vectors have shown efficacy, although they also have shortcomings. PEIs have decent DNA-binding capability and good cell internalization performance, but they cannot deliver gene payloads very efficiently to cell nuclei. In this study, three hyperbranched polyglycerol-polyethylenimine (PG6-PEI) polymers conjugated with myo-inositol (INO) molecules were developed. The three resulting PG6-PEI-INO polymers have an increased number of INO ligands per molecule. PG6-PEI-INO 1 had only 14 carboxymethyl INO (CMINO) units per molecule. PG6-PEI-INO 2 had approximately 130 CMINO units per molecule. PG6-PEI-INO 3 had as high as 415 CMINO units approximately. Mixing PG6-PEI-INO polymers with DNA produced compact nanocomposites. We then performed localization studies using fluorescent microscopy. As the number of conjugated inositol ligands increased in PG6-PEI-INO polymers, there was a corresponding increase in accumulation of the polymers within 293T cell nuclei. Transfection performed with spherical 293T cells yielded 82% of EGFP-positive cells when using PG6-PEI-INO 3 as the vehicle. Studies further revealed that extracellular adenosine triphosphate (eATP) can inhibit the transgene efficiency of PG6-PEI-INO polymers, as compared with PEI and PG6-PEI that were not conjugated with inositol. Our work unveiled the possibility of using inositol as an effective ligand for transgene expression. PMID:25926732

  17. Efficient pseudo-enantiomeric carbohydrate olefin ligands.

    PubMed

    Grugel, Holger; Albrecht, Fabian; Minuth, Tobias; Boysen, Mike M K

    2012-07-20

    Highly efficient pseudo-enantiomeric olefin ligands were designed from D-glucose and D-galactose. These ligands yield consistently excellent levels of enantioselectivity in Rh(I)-catalyzed 1,4-additions of aryl- and alkenylboronic acids to achiral enones and high diastereoselectivity with chiral substrates. Contrary to established olefin ligands, they are obtained enantiomerically pure via short syntheses without racemic resolution steps, making them a valuable addition to the arsenal of chiral ligands with olefinic donor sites. PMID:22780685

  18. Multiplexed biomarker detection using x-ray fluorescence of composition-encoded nanoparticles

    SciTech Connect

    Hossain, Mainul; Wang Chaoming; Su Ming

    2010-12-27

    Multiple DNA and protein biomarkers have been detected based on characteristic x-ray fluorescence of a panel of metal and alloy nanoparticles, which are modified with ligands of biomarkers to create a one-to-one correspondence and immobilized on ligand-modified substrates after forming complexes with target biomarkers in three-strand or sandwich configuration. By determining the presence and concentration of nanoparticles using x-ray fluorescence, the nature and amount of biomarkers can be detected with limits of 1 nM for DNA and 1 ng/ml for protein. By combining high penetrating ability of x-rays, this method allows quantitative imaging of multiple biomarkers.

  19. Combining quantum mechanical ligand conformation analysis and protein modeling to elucidate GPCR-ligand binding modes.

    PubMed

    Schultes, Sabine; Engelhardt, Harald; Roumen, Luc; Zuiderveld, Obbe P; Haaksma, Eric E J; de Esch, Iwan J P; Leurs, Rob; de Graaf, Chris

    2013-01-01

    SAR beyond protein-ligand interactions: By combining structure-affinity relationships, protein-ligand modeling studies, and quantum mechanical calculations, we show that ligand conformational energies and basicity play critical roles in ligand binding to the histamine H4 receptor, a GPCR that plays a key role in inflammation. PMID:23161844

  20. Non-Covalent Fluorescent Labeling of Hairpin DNA Probe Coupled with Hybridization Chain Reaction for Sensitive DNA Detection.

    PubMed

    Song, Luna; Zhang, Yonghua; Li, Junling; Gao, Qiang; Qi, Honglan; Zhang, Chengxiao

    2016-04-01

    An enzyme-free signal amplification-based assay for DNA detection was developed using fluorescent hairpin DNA probes coupled with hybridization chain reaction (HCR). The hairpin DNAs were designed to contain abasic sites in the stem moiety. Non-covalent labeling of the hairpin DNAs was achieved when a fluorescent ligand was bound to the abasic sites through hydrogen bonding with the orphan cytosine present on the complementary strand, accompanied by quench of ligand fluorescence. As a result, the resultant probes, the complex formed between the hairpin DNA and ligand, showed almost no fluorescence. Upon hybridization with target DNA, the probe underwent a dehybridization of the stem moiety containing an abasic site. The release of ligand from the abasic site to the solution resulted in an effective fluorescent enhancement, which can be used as a signal. Compared with a sensing system without HCR, a 20-fold increase in the sensitivity was achieved using the sensing system with HCR. The fluorescent intensity of the sensing system increased with the increase in target DNA concentration from 0.5 nM to 100 nM. A single mismatched target ss-DNA could be effectively discriminated from complementary target DNA. Genotyping of a G/C single-nucleotide polymorphism of polymerase chain reaction (PCR) products was successfully demonstrated with the sensing system. Therefore, integrating HCR strategy with non-covalent labeling of fluorescent hairpin DNA probes provides a sensitive and cost-effective DNA assay. PMID:26879193

  1. Ligand chain length conveys thermochromism.

    PubMed

    Ganguly, Mainak; Panigrahi, Sudipa; Chandrakumar, K R S; Sasmal, Anup Kumar; Pal, Anjali; Pal, Tarasankar

    2014-08-14

    Thermochromic properties of a series of non-ionic copper compounds have been reported. Herein, we demonstrate that Cu(II) ion with straight-chain primary amine (A) and alpha-linolenic (fatty acid, AL) co-jointly exhibit thermochromic properties. In the current case, we determined that thermochromism becomes ligand chain length-dependent and at least one of the ligands (A or AL) must be long chain. Thermochromism is attributed to a balanced competition between the fatty acids and amines for the copper(II) centre. The structure-property relationship of the non-ionic copper compounds Cu(AL)2(A)2 has been substantiated by various physical measurements along with detailed theoretical studies based on time-dependent density functional theory. It is presumed from our results that the compound would be a useful material for temperature-sensor applications. PMID:24943491

  2. Fluorescence in insects

    NASA Astrophysics Data System (ADS)

    Welch, Victoria L.; Van Hooijdonk, Eloise; Intrater, Nurit; Vigneron, Jean-Pol

    2012-10-01

    Fluorescent molecules are much in demand for biosensors, solar cells, LEDs and VCSEL diodes, therefore, considerable efforts have been expended in designing and tailoring fluorescence to specific technical applications. However, naturally occurring fluorescence of diverse types has been reported from a wide array of living organisms: most famously, the jellyfish Aequorea victoria, but also in over 100 species of coral and in the cuticle of scorpions, where it is the rule, rather than the exception. Despite the plethora of known insect species, comparatively few quantitative studies have been made of insect fluorescence. Because of the potential applications of natural fluorescence, studies in this field have relevance to both physics and biology. Therefore, in this paper, we review the literature on insect fluorescence, before documenting its occurrence in the longhorn beetles Sternotomis virescens, Sternotomis variabilis var. semi rufescens, Anoplophora elegans and Stellognatha maculata, the tiger beetles Cicindela maritima and Cicindela germanica and the weevil Pachyrrhynchus gemmatus purpureus. Optical features of insect fluorescence, including emitted wavelength, molecular ageing and naturally occurring combinations of fluorescence with bioluminescence and colour-producing structures are discussed.

  3. A Novel Physical Approach for Cationic–Thiolate Protected Fluorescent Gold Nanoparticles

    PubMed Central

    Ishida, Yohei; Lee, Chaiyathat; Yonezawa, Tetsu

    2015-01-01

    Knowledge on the synthesis of cationically charged fluorescent gold nanoparticles (Au NPs) is limited because the electrostatic repulsion between cationic ligands on the surface of NP hinders the formation of small Au NPs (usually less than ca. 2 nm) during nucleation in solvents. We herein propose a novel methodology for a synthesis of water-dispersible, cationic–thiolate protected fluorescent Au NPs by the sputtering of Au into liquid matrix containing thiolate ligands. By controlling mercaptan concentration the size and photophysical characteristics of Au NPs were directly controlled, resulting in near IR fluorescence with a 0.9% of absolute quantum yield. Cationically charged fluorescent metal NPs are promising, especially in biological fields, and this work provides a novel methodology towards the synthesis of a new series of functional metal NPs. PMID:26482644

  4. Disassembly mediated fluorescence recovery of gold nanodots for selective sulfide sensing

    NASA Astrophysics Data System (ADS)

    Yuan, Zhiqin; Peng, Meihua; Shi, Lei; Du, Yi; Cai, Na; He, Yan; Chang, Huan-Tsung; Yeung, Edward S.

    2013-05-01

    We report a one-pot, two-step strategy to synthesize fluorescent gold nanodots (AuNDs) co-modified with 1-(10-mercaptodecyl)-5-methylpyrimidine-2,4-dione (TSH) and 11-mercaptoundecanoic acid (MUA) through a ligand exchange reaction and demonstrate their capability of selective sulfide sensing in aqueous media on the basis of fluorescence recovery.We report a one-pot, two-step strategy to synthesize fluorescent gold nanodots (AuNDs) co-modified with 1-(10-mercaptodecyl)-5-methylpyrimidine-2,4-dione (TSH) and 11-mercaptoundecanoic acid (MUA) through a ligand exchange reaction and demonstrate their capability of selective sulfide sensing in aqueous media on the basis of fluorescence recovery. Electronic supplementary information (ESI) available: Experimental details, more characterization and other supporting data. See DOI: 10.1039/c2nr33202g

  5. Mg(+)-ligand binding energies

    NASA Technical Reports Server (NTRS)

    Bauschlicher, Charles W., Jr.; Partridge, Harry

    1991-01-01

    Ab initio calculations are used to optimize the structures and determine the binding energies of Mg(+) to a series of ligands. Mg(+) bonds electrostatically with benzene, acetone, H2, CO, and NH3 and a self-consistent-field treatment gives a good description of the bonding. The bonding in MgCN(+) and MgCH3(+) is largely covalent and a correlated treatment is required.

  6. Unusual ligand coordination for cesium

    SciTech Connect

    Bryan, J.C.; Kavallieratos, K.; Sachleben, R.A.

    2000-04-03

    When complexed by tetrabenzo-24-crown-8, the cesium ion can accommodate unprecedented ligation. The structures of the complexes are presented. These structures are the first reported examples of linear {eta}{sup 2}-acetonitrile coordination to any metal ion and the first structures illustrating {eta}{sup 2}-acetonitrile and dichloromethane ligation to an alkali metal ion. Possible steric and electronic origins of these unusual metal-ligand interactions are discussed.

  7. Presentation of Ligands on Hydroxylapatite

    NASA Technical Reports Server (NTRS)

    Chu, Barbara C. F.; Orgel, Leslie E.

    1997-01-01

    Conjugates of biotin with the decamer of glutamic acid (glu(sub 10)) and the trimer of D,L-2-amino-5-phosphonovaleric acid (I) have been synthesized, and it has been shown that they mediate the binding of avidin to hydroxylapatite. In a similar way a conjugate of methotrexate with glu(sub 10) mediates the binding of dihydrofolate reductase to the mineral. The presentation of ligands on the hydroxylapatite component of bone may find applications in clinical medicine.

  8. Absolute Ligand Discrimination by Dimeric Signaling Receptors.

    PubMed

    Fathi, Sepehr; Nayak, Chitra R; Feld, Jordan J; Zilman, Anton G

    2016-09-01

    Many signaling pathways act through shared components, where different ligand molecules bind the same receptors or activate overlapping sets of response regulators downstream. Nevertheless, different ligands acting through cross-wired pathways often lead to different outcomes in terms of the target cell behavior and function. Although a number of mechanisms have been proposed, it still largely remains unclear how cells can reliably discriminate different molecular ligands under such circumstances. Here we show that signaling via ligand-induced receptor dimerization-a very common motif in cellular signaling-naturally incorporates a mechanism for the discrimination of ligands acting through the same receptor. PMID:27602720

  9. Bifunctional crosslinking ligands for transthyretin

    PubMed Central

    Mangione, P. Patrizia; Deroo, Stéphanie; Ellmerich, Stephan; Bellotti, Vittorio; Kolstoe, Simon; Wood, Stephen P.; Robinson, Carol V.; Smith, Martin D.; Tennent, Glenys A.; Broadbridge, Robert J.; Council, Claire E.; Thurston, Joanne R.; Steadman, Victoria A.; Vong, Antonio K.; Swain, Christopher J.; Pepys, Mark B.; Taylor, Graham W.

    2015-01-01

    Wild-type and variant forms of transthyretin (TTR), a normal plasma protein, are amyloidogenic and can be deposited in the tissues as amyloid fibrils causing acquired and hereditary systemic TTR amyloidosis, a debilitating and usually fatal disease. Reduction in the abundance of amyloid fibril precursor proteins arrests amyloid deposition and halts disease progression in all forms of amyloidosis including TTR type. Our previous demonstration that circulating serum amyloid P component (SAP) is efficiently depleted by administration of a specific small molecule ligand compound, that non-covalently crosslinks pairs of SAP molecules, suggested that TTR may be also amenable to this approach. We first confirmed that chemically crosslinked human TTR is rapidly cleared from the circulation in mice. In order to crosslink pairs of TTR molecules, promote their accelerated clearance and thus therapeutically deplete plasma TTR, we prepared a range of bivalent specific ligands for the thyroxine binding sites of TTR. Non-covalently bound human TTR–ligand complexes were formed that were stable in vitro and in vivo, but they were not cleared from the plasma of mice in vivo more rapidly than native uncomplexed TTR. Therapeutic depletion of circulating TTR will require additional mechanisms. PMID:26400472

  10. Tumor Targeting via Integrin Ligands

    PubMed Central

    Marelli, Udaya Kiran; Rechenmacher, Florian; Sobahi, Tariq Rashad Ali; Mas-Moruno, Carlos; Kessler, Horst

    2013-01-01

    Selective and targeted delivery of drugs to tumors is a major challenge for an effective cancer therapy and also to overcome the side-effects associated with current treatments. Overexpression of various receptors on tumor cells is a characteristic structural and biochemical aspect of tumors and distinguishes them from physiologically normal cells. This abnormal feature is therefore suitable for selectively directing anticancer molecules to tumors by using ligands that can preferentially recognize such receptors. Several subtypes of integrin receptors that are crucial for cell adhesion, cell signaling, cell viability, and motility have been shown to have an upregulated expression on cancer cells. Thus, ligands that recognize specific integrin subtypes represent excellent candidates to be conjugated to drugs or drug carrier systems and be targeted to tumors. In this regard, integrins recognizing the RGD cell adhesive sequence have been extensively targeted for tumor-specific drug delivery. Here we review key recent examples on the presentation of RGD-based integrin ligands by means of distinct drug-delivery systems, and discuss the prospects of such therapies to specifically target tumor cells. PMID:24010121

  11. Synthesis, structure and fluorescence properties of a novel 3D Sr(II) coordination polymer

    NASA Astrophysics Data System (ADS)

    Tan, Yu-Hui; Xu, Qing; Gu, Zhi-Feng; Gao, Ji-Xing; Wang, Bin; Liu, Yi; Yang, Chang-Shan; Tang, Yun-Zhi

    2016-09-01

    Solvothermal reaction of 2,2‧-bipyridine-5,5‧-dicarboxylic acid (H2bpdc) and SrCl2 affords a novel coordination polymer [Sr(Hbpdc)2]n1. X-ray structure determination shows that 1 exhibits a novel three-dimensional network. The unique Sr II cation sits on a two-fold axis and coordinated by four O-atom donors from four Hbptc- ligands and four N-atom donors from two Hbptc- ligands in distorted dodecahedral geometry. In 1 each Sr II cation connects to six different Hbptc- ligands and each Hbptc- ligand bridges three different Sr II cations which results in the formation of a three-dimensional polymeric structure. Corresponding to the free ligand, the fluorescent emission of complex 1 display remarkable "Einstain" shifts, which may be attributed to the coordination interaction of Sr atoms, thus reduce the rigidity of pyridyl rings.

  12. Effects of surface ligands and solvents on quantum dot photostability under pulsed UV laser irradiation

    NASA Astrophysics Data System (ADS)

    Krivenkov, Victor A.; Samokhvalov, Pavel S.; Linkov, Pavel A.; Prokhorov, Sergey D.; Martynov, Igor L.; Chistyakov, Alexander A.; Nabiev, Igor

    2015-05-01

    The organic ligands passivating the surface of semiconductor quantum dots (QDs) and the solvents used strongly determine the photostability of QD solutions. Highly purified QD solutions in chloroform have been shown to photodegrade upon pulsed ultraviolet (UV) irradiation, irrespectively of the type of surface ligand. However, the photostability of QDs dissolved in n-octane, a more photochemically inert solvent, strongly depends on the ligands passivating their surface. In n-octane, hexadecylamine-coated QDs are completely stable and display no photochemical response to pulsed UV laser irradiation. In solutions of octanethiol-capped QDs, the photoluminescence intensity slightly decreases under irradiation. QDs coated with trioctylphosphine oxide exhibit a more complex pattern of photobleaching, which depends on the initial value of fluorescence quantum yield of QDs. This complex pattern may be accounted for by two competing processes: (1) ligand photodesorption accompanied by photobleaching due to specific alignment of the band levels of QDs and highest occupied molecular orbital of the ligand and (2) photoinduced decrease in the population of trapping states. Furthermore, practically no thermodynamic degradation of QD solutions has been observed for the micromolar QD concentration used in the study, in contrast to lower concentrations, thus confirming the photoinduced origin of the changes caused by UV irradiation. Obtained results show that the photostability of QDs may be strongly increased by careful selection of the ligands passivating their surface and the solvents used in the experiments.

  13. Evaluation of Polymeric Nanomedicines Targeted to PSMA: Effect of Ligand on Targeting Efficiency.

    PubMed

    Fuchs, Adrian V; Tse, Brian W C; Pearce, Amanda K; Yeh, Mei-Chun; Fletcher, Nicholas L; Huang, Steve S; Heston, Warren D; Whittaker, Andrew K; Russell, Pamela J; Thurecht, Kristofer J

    2015-10-12

    Targeted nanomedicines offer a strategy for greatly enhancing accumulation of a therapeutic within a specific tissue in animals. In this study, we report on the comparative targeting efficiency toward prostate-specific membrane antigen (PSMA) of a number of different ligands that are covalently attached by the same chemistry to a polymeric nanocarrier. The targeting ligands included a small molecule (glutamate urea), a peptide ligand, and a monoclonal antibody (J591). A hyperbranched polymer (HBP) was utilized as the nanocarrier and contained a fluorophore for tracking/analysis, whereas the pendant functional chain-ends provided a handle for ligand conjugation. Targeting efficiency of each ligand was assessed in vitro using flow cytometry and confocal microscopy to compare degree of binding and internalization of the HBPs by human prostate cancer (PCa) cell lines with different PSMA expression status (PC3-PIP (PSMA+) and PC3-FLU (PSMA-). The peptide ligand was further investigated in vivo, in which BALB/c nude mice bearing subcutaneous PC3-PIP and PC3-FLU PCa tumors were injected intravenously with the HBP-peptide conjugate and assessed by fluorescence imaging. Enhanced accumulation in the tumor tissue of PC3-PIP compared to PC3-FLU highlighted the applicability of this system as a future imaging and therapeutic delivery vehicle. PMID:26335533

  14. Ligand identification using electron-density mapcorrelations

    SciTech Connect

    Terwilliger, Thomas C.; Adams, Paul D.; Moriarty, Nigel W.; Cohn,Judith D.

    2006-12-01

    A procedure for the identification of ligands bound incrystal structuresof macromolecules is described. Two characteristics ofthe density corresponding to a ligand are used in the identificationprocedure. One is the correlation of the ligand density with each of aset of test ligands after optimization of the fit of that ligand to thedensity. The other is the correlation of a fingerprint of the densitywith the fingerprint of model density for each possible ligand. Thefingerprints consist of an ordered list of correlations of each the testligands with the density. The two characteristics are scored using aZ-score approach in which the correlations are normalized to the mean andstandard deviation of correlations found for a variety of mismatchedligand-density pairs, so that the Z scores are related to the probabilityof observing a particular value of the correlation by chance. Theprocedure was tested with a set of 200 of the most commonly found ligandsin the Protein Data Bank, collectively representing 57 percent of allligands in the Protein Data Bank. Using a combination of these twocharacteristics of ligand density, ranked lists of ligand identificationswere made for representative (F-o-F-c) exp(i phi(c)) difference densityfrom entries in the Protein Data Bank. In 48 percent of the 200 cases,the correct ligand was at the top of the ranked list of ligands. Thisapproach may be useful in identification of unknown ligands in newmacromolecular structures as well as in the identification of whichligands in a mixture have bound to a macromolecule.

  15. NKG2D ligands as therapeutic targets

    PubMed Central

    Spear, Paul; Wu, Ming-Ru; Sentman, Marie-Louise; Sentman, Charles L.

    2013-01-01

    The Natural Killer Group 2D (NKG2D) receptor plays an important role in protecting the host from infections and cancer. By recognizing ligands induced on infected or tumor cells, NKG2D modulates lymphocyte activation and promotes immunity to eliminate ligand-expressing cells. Because these ligands are not widely expressed on healthy adult tissue, NKG2D ligands may present a useful target for immunotherapeutic approaches in cancer. Novel therapies targeting NKG2D ligands for the treatment of cancer have shown preclinical success and are poised to enter into clinical trials. In this review, the NKG2D receptor and its ligands are discussed in the context of cancer, infection, and autoimmunity. In addition, therapies targeting NKG2D ligands in cancer are also reviewed. PMID:23833565

  16. Characterizing new fluorescent tools for studying 5-HT₃ receptor pharmacology.

    PubMed

    Jack, Thomas; Simonin, Jonathan; Ruepp, Marc-David; Thompson, Andrew J; Gertsch, Jürg; Lochner, Martin

    2015-03-01

    The pharmacological characterization of ligands depends upon the ability to accurately measure their binding properties. Fluorescence provides an alternative to more traditional approaches such as radioligand binding. Here we describe the binding and spectroscopic properties of eight fluorescent 5-HT3 receptor ligands. These were tested on purified receptors, expressed receptors on live cells, or in vivo. All compounds had nanomolar affinities with fluorescent properties extending from blue to near infra-red emission. A fluorescein-derivative had the highest affinity as measured by fluorescence polarization (FP; 1.14 nM), flow cytometry (FC; 3.23 nM) and radioligand binding (RB; 1.90 nM). Competition binding with unlabeled 5-HT3 receptor agonists (5-HT, mCPBG, quipazine) and antagonists (granisetron, palonosetron, tropisetron) yielded similar affinities in all three assays. When cysteine substitutions were introduced into the 5-HT3 receptor binding site the same changes in binding affinity were seen for both granisetron and the fluorescein-derivative, suggesting that they both adopt orientations that are consistent with co-crystal structures of granisetron with a homologous protein (5HTBP). As expected, in vivo live imaging in anaesthetized mice revealed staining in the abdominal cavity in intestines, but also in salivary glands. The unexpected presence of 5-HT3 receptors in mouse salivary glands was confirmed by Western blots. Overall, these results demonstrate the wide utility of our new high-affinity fluorescently-labeled 5-HT3 receptor probes, ranging from in vitro receptor pharmacology, including FC and FP ligand competition, to live imaging of 5-HT3 expressing tissues. PMID:25460187

  17. Dinuclear Zinc (II) Complexes of Macrocyclic Polyamine Ligands Containing an Imidazolium Bridge: Synthesis, Characterization, and Their Interaction with Plasmid DNA

    PubMed Central

    Huang, Jun; Huang, Qing-Dong; Zhang, Ji; Zhou, Li-Hong; Li, Qiang-Lin; Li, Kun; Jiang, Ning; Lin, Hong-Hui; Wu, Jiang; Yu, Xiao-Qi

    2007-01-01

    Two novel macrocyclic polyamine ligands and their dinuclear zinc (II) complexes were synthesized and characterized. Their interaction with plasmid DNA was studied by gel electrophoresis and fluorescence quenching experiment. The result showed that these complexes could bind DNA efficiently under physiological conditions.

  18. Complementary Spectroscopic Assays for Investigating Protein-Ligand Binding Activity: A Project for the Advanced Chemistry Laboratory

    ERIC Educational Resources Information Center

    Mascotti, David P.; Waner, Mark J.

    2010-01-01

    A protein-ligand binding, guided-inquiry laboratory project with potential application across the advanced undergraduate curriculum is described. At the heart of the project are fluorescence and spectrophotometric assays utilizing biotin-4-fluorescein and streptavidin. The use of the same stock solutions for an assay that may be examined by two…

  19. Biophysical characterization of G-protein coupled receptor-peptide ligand binding

    PubMed Central

    Langelaan, David N.; Ngweniform, Pascaline; Rainey, Jan K.

    2011-01-01

    G-protein coupled receptors (GPCRs) are ubiquitous membrane proteins allowing intracellular response to extracellular factors that range from photons of light to small molecules to proteins. Despite extensive exploitation of GRCRs as therapeutic targets, biophysical characterization of GPCR-ligand interactions remains challenging. In this minireview, we focus on techniques which have been successfully employed for structural and biophysical characterization of peptide ligands binding to their cognate GPCRs. The techniques reviewed include solution-state nuclear magnetic resonance (NMR) spectroscopy; solid-state NMR; X-ray diffraction; fluorescence spectroscopy and single molecule fluorescence methods; flow cytometry; surface plasmon resonance; isothermal titration calorimetry; and, atomic force microscopy. The goal herein is to provide a cohesive starting point to allow selection of techniques appropriate to the elucidation of a given GPCR-peptide interaction. PMID:21455262

  20. Metal nanoparticles functionalized with metal-ligand covalent bonds

    NASA Astrophysics Data System (ADS)

    Kang, Xiongwu

    Metal-organic contact has been recognized to play important roles in regulation of optical and electronic properties of nanoparticles. In this thesis, significant efforts have been devoted into synthesis of ruthenium nanoparticles with various metal-ligand interfacial linkages and investigation of their electronic and optical properties. Ruthenium nanoparticles were prepared by the self-assembly of functional group onto bare Ru colloid surface. As to Ru-alkyne nanoparticles, the formation of a Ru-vinylidene (Ru=C=CH--R) interfacial bonding linkage was confirmed by the specific reactivity of the nanoparticles with imine derivatives and olefin at the metal-ligand interface, as manifested in NMR, photoluminescence, and electrochemical measurements. Interestingly, it was found the electronic coupling coefficient (beta)for strongly depend upon such metal-ligand interfacial bonding. Next, such metal-ligand interfacial bonding was extended to ruthenium-nitrene pi bonds on ruthenium colloids, which were investigated by XPS. The nanoparticles exhibited a 1:1 atomic ratio of nitrogen to sulfur, consistent with that of sulfonyl nitrene fragments. In addition, the nanoparticle-bound nitrene moieties behaved analogously to azo derivatives, as manifested in UV-vis and fluorescence measurements. Further testimony of the formation of Ru=N interfacial linkages was highlighted in the unique reactivity of the nanoparticles with alkenes by imido transfer. Extensive conjugation between metal-ligand interfacial bond results in remarkable intraparticle charge delocalization on Ru-alkynide nanoparticles, which was manipulated by simple chemical reduction or oxidation. Charging of extra electrons into the nanoparticle cores led to an electron-rich metal core and hence red-shift of the triple bond stretching mode, lower binding energy of sp hybridized C 1s and dimmed fluorescence of nanoparticles. Instead, chemical oxidation resulted in the opposite impacts on these properties. By taking

  1. FRET enhanced fluorescent nanodiamonds.

    PubMed

    Fudala, Rafal; Raut, Sangram; Maliwal, Badri P; Zerda, T W; Gryczynski, Ignacy; Simanek, Eric; Borejdo, Julian; Rich, Ryan; Akopova, Irina; Gryczynski, Zygmunt

    2014-01-01

    Fluorescent nanodiamonds (FNDs) are one of the new and very promising biocompatible nanomaterials that can be used both as a fluorescence imaging agent and a highly versatile platform for controlled functionalization to target and deliver a wide spectrum of therapeutic agents. Among the remarkable fluorescence properties are excellent photostability, emission between 600-700nm, quantum yield of 1 and moderately long fluorescence lifetimes. However the low absorption cross section of fluorescent (N-V)(-) centers limits FNDs' brightness. In this work we show that an approach based on the Forster resonance energy transfer (FRET) may significantly enhance the fluorescence signal observed from a single ND. We demonstrate that organic dyes (fluorophores) attached to the FND surface can efficiently transfer the excitation energy to (N-V)(-) centers. Multiple dyes positioned in close proximity to the ND facile surface may serve as harvesting antennas transferring excitation energy to the fluorescent centers. We propose that, with the help of some of the functional groups present on the FND surface, we can either directly link flurophores or use scalable dendrimer chemistry to position many organic dyes at a calibrated distance. Also, the remaining multiple functional groups will be still available for particle targeting and drug delivery. This opens a new way for designing a new type of theranostics particles of ultrahigh brightness, high photostability, specific targeting, and high capacity for drug delivery. PMID:22394126

  2. Development of a cyclometalated iridium complex with specific intramolecular hydrogen-bonding that acts as a fluorescent marker for the endoplasmic reticulum and causes photoinduced cell death† †Electronic supplementary information (ESI) available: Detailed synthesis; photophysical data (Table S1); pH dependent phosphorescence lifetime of complex C2 (Table S2); crystallographic parameters of C2; selected bond distances and angles of C2 (Table S3); cyclic voltammetric data of complexes C1–C11 (Table S4); 1H NMR spectra of ligands and complexes (Fig. S1 and S3); ESI-MS spectra of ligands and complexes (Fig. S2 and S4); fluorescence spectra of the complexes in acetonitrile and at pH 4, 7 and 9, exponential decay curve of C2 (Fig. S5); pH dependent fluorescence spectrum of complexes C1–C11 (Fig. S6); DIC and confocal fluorescence images of live MCF7 cells not treated with C2 but exposed to photoirradiation at 405 nm for 30 min; the cells were treated with DCFDA and fluorescence images were obtained at 529 nm after excitation at 495 nm (Fig. S7). ESI videos 1 and 2. CCDC 967841. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c4dt00845f Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.

    PubMed Central

    Mandal, Soumik; Poria, Dipak K.; Ghosh, Ritabrata

    2014-01-01

    Cyclometalated iridium complexes have important applications as phosphorescent probes for cellular imaging due to their photophysical properties. Moreover, these properties also make them potential candidates as photosensitizers for photodynamic therapy (PDT) of tumors and skin diseases. Treatment of MCF7 breast carcinoma cells with a heteroleptic phosphorescent cyclometalated iridium(iii) complex C2 followed by confocal imaging indicates that the complex selectively localizes and exhibits high fluorescence in the endoplasmic reticulum. In an unprecedented approach, systematic alteration of functional groups or the metal core in C2 to synthesize a series of iridium(iii) complexes (C1–C10) and an organometallic rhenium complex C11 with an imidazolyl modified phenanthroline ligand has indicated the functional groups and their interactions that are responsible for this selective localization. Remarkably, the exposure of the cells treated with C2 to irradiation at 405 nm for one hour led to membrane blebbing and cell death, demonstrating a photosensitizing property of the compound. PMID:25341053

  3. Study on the fluorescent chemosensors based on a series of bis-Schiff bases for the detection of zinc(II).

    PubMed

    Wang, Wanguan; Li, Rong; Song, Tianwen; Zhang, Chunjiao; Zhao, Yu

    2016-07-01

    In order to study the influence of different substituent groups on the fluorescence properties, a series of bis-Schiff bases (L) with electron-donating groups (salicylaldehyde, o-vanillin, 2,4-dihydroxybenzaldehyde) and electron-drawing group (4-formylbenzoic acid) have been synthesized, and characterized by IR spectrum, NMR, mass spectrum, and fluorescence spectroscopy. The investigation of the fluorescent properties reveals that the fluorescence can be enhanced when the bis-Schiff base ligands with electron-donating groups complex with Zn ion, while other kinds of metal complexes with these ligands do not show any enhancement, whereas no fluorescence enhancement can be observed when the ligand with electron-drawing group complexes with all different types of metal ions. In addition, as for the ligands with electron-donating groups detecting zinc ion, the fluorescence intensity is linear correlated with the concentration of zinc ion. Therefore, the study indicates that the ligands with electron-donating groups can be used as Zn ion fluorescent sensor. PMID:27092737

  4. Study on the fluorescent chemosensors based on a series of bis-Schiff bases for the detection of zinc(II)

    NASA Astrophysics Data System (ADS)

    Wang, Wanguan; Li, Rong; Song, Tianwen; Zhang, Chunjiao; Zhao, Yu

    2016-07-01

    In order to study the influence of different substituent groups on the fluorescence properties, a series of bis-Schiff bases (L) with electron-donating groups (salicylaldehyde, o-vanillin, 2,4-dihydroxybenzaldehyde) and electron-drawing group (4-formylbenzoic acid) have been synthesized, and characterized by IR spectrum, NMR, mass spectrum, and fluorescence spectroscopy. The investigation of the fluorescent properties reveals that the fluorescence can be enhanced when the bis-Schiff base ligands with electron-donating groups complex with Zn ion, while other kinds of metal complexes with these ligands do not show any enhancement, whereas no fluorescence enhancement can be observed when the ligand with electron-drawing group complexes with all different types of metal ions. In addition, as for the ligands with electron-donating groups detecting zinc ion, the fluorescence intensity is linear correlated with the concentration of zinc ion. Therefore, the study indicates that the ligands with electron-donating groups can be used as Zn ion fluorescent sensor.

  5. Chemistry and pharmacology of GABAB receptor ligands.

    PubMed

    Froestl, Wolfgang

    2010-01-01

    This chapter presents new clinical applications of the prototypic GABA(B) receptor agonist baclofen for the treatment of addiction by drugs of abuse, such as alcohol, cocaine, nicotine, morphine, and heroin, a novel baclofen prodrug Arbaclofen placarbil, the GABA(B) receptor agonist AZD3355 (Lesogabaran) currently in Phase 2 clinical trials for the treatment of gastroesophageal reflux disease, and four positive allosteric modulators of GABA(B) receptors (CGP7930, GS39783, NVP-BHF177, and BHFF), which have less propensity for the development of tolerance due to receptor desensitization than classical GABA(B) receptor agonists. All four compounds showed anxiolytic affects. In the presence of positive allosteric modulators the "classical" GABA(B) receptor antagonists CGP35348 and 2-hydroxy-saclofen showed properties of partial GABA(B) receptor agonists. Seven micromolar affinity GABA(B) receptor antagonists, phaclofen; 2-hydroxy-saclofen; CGP's 35348, 36742, 46381, 51176; and SCH50911, are discussed. CGP36742 (SGS742) showed statistically significant improvements of working memory and attention in a Phase 2 clinical trial in mild, but not in moderate Alzheimer patients. Eight nanomolar affinity GABA(B) receptor antagonists are presented (CGP's 52432, 54626, 55845, 56433, 56999, 61334, 62349, and 63360) that were used by pharmacologists for numerous in vitro and in vivo investigations. CGP's 36742, 51176, 55845, and 56433 showed antidepressant effects. Several compounds are also available as radioligands, such as [(3)H]CGP27492, [(3)H]CGP54626, [(3)H]CGP5699, and [(3)H]CGP62349. Three novel fluorescent and three GABA(B) receptor antagonists with very high specific radioactivity (>2,000 Ci/mmol) are presented. [(125)I]CGP64213 and the photoaffinity ligand [(125)I]CGP71872 allowed the identification of GABA(B1a) and GABA(B1b) receptors in the expression cloning work. PMID:20655477

  6. Highly thermostable fluorescent proteins

    DOEpatents

    Bradbury, Andrew M.; Waldo, Geoffrey S.; Kiss, Csaba

    2011-03-22

    Thermostable fluorescent proteins (TSFPs), methods for generating these and other stability-enhanced proteins, polynucleotides encoding such proteins, and assays and method for using the TSFPs and TSFP-encoding nucleic acid molecules are provided. The TSFPs of the invention show extremely enhanced levels of stability and thermotolerance. In one case, for example, a TSFP of the invention is so stable it can be heated to 99.degree. C. for short periods of time without denaturing, and retains 85% of its fluorescence when heated to 80.degree. C. for several minutes. The invention also provides a method for generating stability-enhanced variants of a protein, including but not limited to fluorescent proteins.

  7. Highly thermostable fluorescent proteins

    DOEpatents

    Bradbury, Andrew M.; Waldo, Geoffrey S.; Kiss, Csaba

    2012-05-01

    Thermostable fluorescent proteins (TSFPs), methods for generating these and other stability-enhanced proteins, polynucleotides encoding such proteins, and assays and method for using the TSFPs and TSFP-encoding nucleic acid molecules are provided. The TSFPs of the invention show extremely enhanced levels of stability and thermotolerance. In one case, for example, a TSFP of the invention is so stable it can be heated to 99.degree. C. for short periods of time without denaturing, and retains 85% of its fluorescence when heated to 80.degree. C. for several minutes. The invention also provides a method for generating stability-enhanced variants of a protein, including but not limited to fluorescent proteins.

  8. Highly thermostable fluorescent proteins

    DOEpatents

    Bradbury, Andrew M.; Waldo, Geoffrey S.; Kiss, Csaba

    2011-11-29

    Thermostable fluorescent proteins (TSFPs), methods for generating these and other stability-enhanced proteins, polynucleotides encoding such proteins, and assays and method for using the TSFPs and TSFP-encoding nucleic acid molecules are provided. The TSFPs of the invention show extremely enhanced levels of stability and thermotolerance. In one case, for example, a TSFP of the invention is so stable it can be heated to 99.degree. C. for short periods of time without denaturing, and retains 85% of its fluorescence when heated to 80.degree. C. for several minutes. The invention also provides a method for generating stability-enhanced variants of a protein, including but not limited to fluorescent proteins.

  9. Fluorescent discharge lamp

    NASA Technical Reports Server (NTRS)

    Mukai, E.; Otsuka, H.; Nomi, K.; Honmo, I.

    1982-01-01

    A rapidly illuminating fluorescent lamp 1,200 mm long and 32.5 mm in diameter with an interior conducting strip which is compatible with conventional fixtures and ballasts is described. The fluorescent lamp is composed of a linear glass tube, electrodes sealed at both ends, mercury and raregas sealed in the glass tube, a fluorescent substance clad on the inner walls of the glass tube, and a clad conducting strip extending the entire length of the glass tube in the axial direction on the inner surface of the tube.

  10. UV and sunlight driven photoligation of quantum dots: understanding the photochemical transformation of the ligands.

    PubMed

    Aldeek, Fadi; Hawkins, Dana; Palomo, Valle; Safi, Malak; Palui, Goutam; Dawson, Philip E; Alabugin, Igor; Mattoussi, Hedi

    2015-02-25

    We have recently reported that photoinduced ligation of ZnS-overcoated quantum dots (QDs) offers a promising strategy to promote the phase transfer of these materials to polar and aqueous media using multidentate lipoic acid (LA)-modified ligands. In this study we investigate the importance of the underlying parameters that control this process, in particular, whether or not photoexcited QDs play a direct role in the photoinduced ligation. We find that irradiation of the ligand alone prior to mixing with hydrophobic QDs is sufficient to promote ligand exchange. Furthermore, photoligation onto QDs can also be carried out simply by using sunlight. Combining the use of Ellman's test with matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry, we probe the nature of the photochemical transformation of the ligands. We find that irradiation (using either a UV photoreactor or sunlight) alters the nature of the disulfide groups in the lipoic acid, yielding a different product mixture than what is observed for chemically reduced ligands. Irradiation of the ligand in solution generates a mixture of monomeric and oligomeric compounds. Ligation onto the QDs selectively favors oligomers, presumably due to their higher coordination onto the metal-rich QD surfaces. We also show that photoligation using mixed ligands allows the preparation of reactive nanocrystals. The resulting QDs are coupled to proteins and peptides and tested for cellular staining. This optically controlled ligation of QDs combined with the availability of a variety of multidentate and multifunctional LA-modified ligands open new opportunities for developing fluorescent platforms with great promises for use in imaging and sensor design. PMID:25612193

  11. smFRET studies of the ‘encounter’ complexes and subsequent intermediate states that regulate the selectivity of ligand binding

    PubMed Central

    Kinz-Thompson, Colin D.; Gonzalez, Ruben L.

    2016-01-01

    The selectivity with which a biomolecule can bind its cognate ligand when confronted by the vast array of structurally similar, competing ligands that are present in the cell underlies the fidelity of some of the most fundamental processes in biology. Because they collectively comprise one of only a few methods that can sensitively detect the ‘encounter’ complexes and subsequent intermediate states that regulate the selectivity of ligand binding, single-molecule fluorescence, and particularly single-molecule fluorescence resonance energy transfer (smFRET), approaches have revolutionized studies of ligand-binding reactions. Here, we describe a widely used smFRET strategy that enables investigations of a large variety of ligand-binding reactions, and discuss two such reactions, aminoacyl-tRNA selection during translation elongation and splice site selection during spliceosome assembly, that highlight both the successes and challenges of smFRET studies of ligand-binding reactions. We conclude by reviewing a number of emerging experimental and computational approaches that are expanding the capabilities of smFRET approaches for studies of ligand-binding reactions and that promise to reveal the mechanisms that control the selectivity of ligand binding with unprecedented resolution. PMID:25066296

  12. Fluorescent probes for insect ryanodine receptors: candidate anthranilic diamides.

    PubMed

    Wang, Yi; Guo, Lei; Qi, Suzhen; Zhang, Hao; Liu, Kechang; Liu, Ruiquan; Liang, Pei; Casida, John E; Liu, Shangzhong

    2014-01-01

    Diamide insecticides with high efficacy against pests and good environmental safety are broadly applied in crop protection. They act at a poorly-defined site in the very complex ryanodine (Ry) receptor (RyR) potentially accessible to a fluorescent probe. Two N-propynyl analogs of the major anthranilic diamide insecticides chlorantraniliprole (Chlo) and cyantraniliprole (Cyan) were accordingly synthesized and converted into two fluorescent ligands by click reaction coupling with 3-azido-7-hydroxy-2H-chromen-2-one. The new diamide analogs and fluorescent ligands were shown to be nearly as potent as Chlo and Cyan in inhibition of [3H]Chlo binding and stimulation of [3H]Ry binding in house fly thoracic muscle RyR. Although the newly synthesized compounds had only moderate activity in insect larvicidal activity assays, their high in vitro potency in a validated insect RyR binding assay encourages further development of fluorescent probes for insect RyRs. PMID:24699151

  13. Canonical and non-canonical Notch ligands

    PubMed Central

    D’SOUZA, BRENDAN; MELOTY-KAPELLA, LAURENCE; WEINMASTER, GERRY

    2015-01-01

    Notch signaling induced by canonical Notch ligands is critical for normal embryonic development and tissue homeostasis through the regulation of a variety of cell fate decisions and cellular processes. Activation of Notch signaling is normally tightly controlled by direct interactions with ligand-expressing cells and dysregulated Notch signaling is associated with developmental abnormalities and cancer. While canonical Notch ligands are responsible for the majority of Notch signaling, a diverse group of structurally unrelated non-canonical ligands has also been identified that activate Notch and likely contribute to the pleiotropic effects of Notch signaling. Soluble forms of both canonical and non-canonical ligands have been isolated, some of which block Notch signaling and could serve as natural inhibitors of this pathway. Ligand activity can also be indirectly regulated by other signaling pathways at the level of ligand expression, serving to spatio-temporally compartmentalize Notch signaling activity and integrate Notch signaling into a molecular network that orchestrates developmental events. Here, we review the molecular mechanisms underlying the dual role of Notch ligands as activators and inhibitors of Notch signaling. Additionally, evidence that Notch ligands function independent of Notch are presented. We also discuss how ligand post-translational modification, endocytosis, proteolysis and spatio-temporal expression regulate their signaling activity. PMID:20816393

  14. Conformational readout of RNA by small ligands

    PubMed Central

    Kligun, Efrat; Mandel-Gutfreund, Yael

    2013-01-01

    RNA molecules have highly versatile structures that can fold into myriad conformations, providing many potential pockets for binding small molecules. The increasing number of available RNA structures, in complex with proteins, small ligands and in free form, enables the design of new therapeutically useful RNA-binding ligands. Here we studied RNA ligand complexes from 10 RNA groups extracted from the protein data bank (PDB), including adaptive and non-adaptive complexes. We analyzed the chemical, physical, structural and conformational properties of binding pockets around the ligand. Comparing the properties of ligand-binding pockets to the properties of computed pockets extracted from all available RNA structures and RNA-protein interfaces, revealed that ligand-binding pockets, mainly the adaptive pockets, are characterized by unique properties, specifically enriched in rare conformations of the nucleobase and the sugar pucker. Further, we demonstrate that nucleotides possessing the rare conformations are preferentially involved in direct interactions with the ligand. Overall, based on our comprehensive analysis of RNA-ligand complexes, we suggest that the unique conformations adopted by RNA nucleotides play an important role in RNA recognition by small ligands. We term the recognition of a binding site by a ligand via the unique RNA conformations “RNA conformational readout.” We propose that “conformational readout” is a general way by which RNA binding pockets are recognized and selected from an ensemble of different RNA states. PMID:23618839

  15. Ligand placement based on prior structures: the guided ligand-replacement method

    SciTech Connect

    Klei, Herbert E.; Moriarty, Nigel W. Echols, Nathaniel; Terwilliger, Thomas C.; Baldwin, Eric T.; Pokross, Matt; Posy, Shana; Adams, Paul D.

    2014-01-01

    A new module, Guided Ligand Replacement (GLR), has been developed in Phenix to increase the ease and success rate of ligand placement when prior protein-ligand complexes are available. The process of iterative structure-based drug design involves the X-ray crystal structure determination of upwards of 100 ligands with the same general scaffold (i.e. chemotype) complexed with very similar, if not identical, protein targets. In conjunction with insights from computational models and assays, this collection of crystal structures is analyzed to improve potency, to achieve better selectivity and to reduce liabilities such as absorption, distribution, metabolism, excretion and toxicology. Current methods for modeling ligands into electron-density maps typically do not utilize information on how similar ligands bound in related structures. Even if the electron density is of sufficient quality and resolution to allow de novo placement, the process can take considerable time as the size, complexity and torsional degrees of freedom of the ligands increase. A new module, Guided Ligand Replacement (GLR), was developed in Phenix to increase the ease and success rate of ligand placement when prior protein–ligand complexes are available. At the heart of GLR is an algorithm based on graph theory that associates atoms in the target ligand with analogous atoms in the reference ligand. Based on this correspondence, a set of coordinates is generated for the target ligand. GLR is especially useful in two situations: (i) modeling a series of large, flexible, complicated or macrocyclic ligands in successive structures and (ii) modeling ligands as part of a refinement pipeline that can automatically select a reference structure. Even in those cases for which no reference structure is available, if there are multiple copies of the bound ligand per asymmetric unit GLR offers an efficient way to complete the model after the first ligand has been placed. In all of these applications, GLR

  16. Synthesis, crystal structures and properties of three new mixed-ligand d{sup 10} metal complexes constructed from pyridinecarboxylate and in situ generated amino-tetrazole ligand

    SciTech Connect

    Liu Dongsheng; Huang, Xihe; Huang Changcang; Huang Gansheng; Chen Jianzhong

    2009-07-15

    Three new metal-organic frameworks, [Zn(atz)(nic)]{sub n}(1), [Zn(atz)(isonic)]{sub n}.nHisonic(2) and [Cd(atz)(isonic)]{sub n}(3) (Hnic=nicotinic acid, Hisonic=isonicotinic acid), have been firstly synthesized by employing mixed-ligand of pyridinecarboxylate with the in situ generated ligand of 5-amino-tetrazolate(atz{sup -}), and characterized by elemental analysis, IR spectroscopy, TGA and single crystal X-ray diffraction. The results revealed that 1 presents a two-dimensional (2D) 'sql' topological network constructed from the linear chain subunit of Zn(nic){sub 2} and atz{sup -} ligand. A remarkable feature of 2 is a 2-fold interpenetrated diamondoid network with free Hisonic molecules locating in the channels formed by the zigzag chain subunits of Zn(isonic){sub 2}. Complex 3 is a 3D non-interpenetrated pillared framework constructed from the double chain subunits of Cd-COO{sup -}Cd. It possesses a rarely observed (4,6)-connected 'fsc' topology. The thermal stabilities and fluorescent properties of the complexes were investigated. All of these complexes exhibited intense fluorescent emissions in the solid state at room temperature. - Graphical abstract: Three new mixed-ligand d{sup 10} metal complexes have been synthesized by employing mixed-ligand synthetic approach. Complex 1 presents a 2D 'sql' topological network. Complex 2 is a 2-fold interpenetrated diamondoid network with microporous channels. Rarely observed (4,6)-connected 'fsc' topological network was found in complex 3.

  17. Ligand enabling visible wavelength excitation of europium(III) for fluoroimmunoassays in aqueous micellar solutions.

    PubMed

    Valta, Timo; Puputti, Eeva-Maija; Hyppänen, Iko; Kankare, Jouko; Takalo, Harri; Soukka, Tero

    2012-09-18

    Fluorescent reporters based on lanthanide ions, such as europium chelates, enable highly sensitive detection in immunoassays and other ligand binding assays. Unfortunately they normally require UV-excitation produced by a xenon flash or nitrogen laser light source. In order to use modern solid state excitation sources such as light emitting diodes (LEDs), these reporters need to be excited at wavelengths longer than 365 nm, where high-powered ultraviolet LEDs are available. A novel ligand, 9-ethyl-3,6-bis(5',5',5',4',4'-pentafluoro-1',3'-dioxopentyl)carbazole (bdc), was synthesized to efficiently excite europium(III) at wavelengths up to 450 nm in micellar solutions, and its performance was compared to a commercially available DELFIA enhancement solution. The detection limit of Eu(III) with the bdc-ligand using 365 nm excitation was determined to be 63 fM, which is 3 times lower than with the DELFIA solution. The bdc-ligand enabled sensitive detection of europium(III) ions in solution using 365 nm excitation and displayed similar sensitivity and functionality as commercially available DELFIA enhancement solution. Therefore, this novel enhancement solution might be a feasible alternative in producing time-resolved fluorescence under LED-excitation. PMID:22901050

  18. Synthesis of functionalized fluorescent gold nanoclusters for acid phosphatase sensing

    NASA Astrophysics Data System (ADS)

    Sun, Jian; Yang, Fan; Yang, Xiurong

    2015-10-01

    A novel and convenient one-pot but two-step synthesis of fluorescent gold nanoclusters, incorporating glutathione (GSH) and 11-mercaptoundecanoic acid (MUA) as the functionalized ligands (i.e. AuNCs@GSH/MUA), is demonstrated. Herein, the mixing of HAuCl4 and GSH in aqueous solution results in the immediate formation of non-fluorescent GSH-Au+ complexes, and then a class of ~2.6 nm GSH-coated AuNCs (AuNCs@GSH) with mild orange-yellow fluorescence after several days. Interestingly, the intense orange-red emitting ~1.7 nm AuNCs@GSH/MUA can be synthesized within seconds by introducing an alkaline aqueous solution of MUA into the GSH-Au+ complexes or AuNC@GSH solution. Subsequently, a reliable AuNC@GSH/MUA-based real-time assay of acid phosphatase (ACP) is established for the first time, inspired by the selective coordination of Fe3+ with surface ligands of AuNCs, the higher binding affinity between the pyrophosphate ion (PPi) and Fe3+, and the hydrolysis of PPi into orthophosphate by ACP. Our fluorescent chemosensor can also be applied to assay ACP in a real biological sample and, furthermore, to screen the inhibitor of ACP. This report paves a new avenue for synthesizing AuNCs based on either the bottom-up reduction or top-down etching method, establishing real-time fluorescence assays for ACP by means of PPi as the substrate, and further exploring the sensing applications of fluorescent AuNCs.A novel and convenient one-pot but two-step synthesis of fluorescent gold nanoclusters, incorporating glutathione (GSH) and 11-mercaptoundecanoic acid (MUA) as the functionalized ligands (i.e. AuNCs@GSH/MUA), is demonstrated. Herein, the mixing of HAuCl4 and GSH in aqueous solution results in the immediate formation of non-fluorescent GSH-Au+ complexes, and then a class of ~2.6 nm GSH-coated AuNCs (AuNCs@GSH) with mild orange-yellow fluorescence after several days. Interestingly, the intense orange-red emitting ~1.7 nm AuNCs@GSH/MUA can be synthesized within seconds by

  19. Aqueous synthesis of near-infrared highly fluorescent platinum nanoclusters

    NASA Astrophysics Data System (ADS)

    García Fernández, Jenifer; Trapiella-Alfonso, Laura; Costa-Fernández, José M.; Pereiro, Rosario; Sanz-Medel, Alfredo

    2015-05-01

    A one-step synthesis of near infrared fluorescent platinum nanoclusters (PtNCs) in aqueous medium is described. The proposed optimized procedure for PtNC synthesis is rather simple, fast and it is based on the direct metal reduction with NaBH4. Bidentated thiol ligands (lipoic acid) were selected as nanoclusters stabilizers in water media. The structural characterization revealed attractive features of the PtNCs, including small size, high water solubility, near-infrared luminescence centered at 680 nm, long-term stability and the highest quantum yield in water reported so far (47%) for PtNCs. Moreover, their stability in different pH media and an ionic strength of 0.2 M NaCl was studied and no significant changes in fluorescence emission were detected. In brief, they offer a new type of fluorescent noble metal nanoprobe with a great potential to be applied in several fields, including biolabeling and imaging experiments.

  20. Fluorescent radiation converter

    NASA Technical Reports Server (NTRS)

    Viehmann, W. (Inventor)

    1981-01-01

    A fluorescence radiation converter is described which includes a substantially undoped optically transparent substrate and a waveshifter coating deposited on at least one portion of the substrate for absorption of radiation and conversion of fluorescent radiation. The coating is formed to substantially 1000 g/liter of a solvent, 70 to 200 g/liter of an organic polymer, and 0.2 to 25 g/liter of at least one organic fluorescent dye. The incoming incident radiation impinges on the coating. Radiation is absorbed by the fluorescent dye and is re-emitted as a longer wavelength radiation. Radiation is trapped within the substrate and is totally internally reflected by the boundary surface. Emitted radiation leaves the substrate ends to be detected.

  1. Fluorescent filtered electrophosphorescence

    DOEpatents

    Forrest, Stephen; Sun, Yiru; Giebink, Noel; Thompson, Mark E.

    2010-08-03

    The present invention relates to organic light emitting devices (OLEDs), and more specifically to OLEDS that emit light using a combination of fluorescent emitters and phosphorescent emitters for the efficient utilization of all of the electrically generated excitons.

  2. Fluorescent filtered electrophosphorescence

    DOEpatents

    Forrest, Stephen R.; Sun, Yiru; Giebink, Noel; Thompson, Mark E.

    2009-01-06

    The present invention relates to organic light emitting devices (OLEDs), and more specifically to OLEDS that emit light using a combination of fluorescent emitters and phosphorescent emitters for the efficient utilization of all of the electrically generated excitons.

  3. Fluorescent eye test (image)

    MedlinePlus

    The fluorescent eye test is useful in determining if there is a scratch or other problem with the surface ... has thoroughly covered the eye a cobalt blue light is then directed on the eye. The light ...

  4. Atmospheric Fluorescence Yield

    NASA Technical Reports Server (NTRS)

    Adams, James H., Jr.; Christl, M. J.; Fountain, W. F.; Gregory, J. C.; Martens, K.; Sokolsky, P.; Whitaker, Ann F. (Technical Monitor)

    2001-01-01

    Several existing and planned experiments estimate the energies of ultra-high energy cosmic rays from air showers using the atmospheric fluorescence from these showers. Accurate knowledge of the conversion from atmospheric fluorescence to energy loss by ionizing particles in the atmosphere is key to this technique. In this paper we discuss a small balloon-borne instrument to make the first in situ measurements versus altitude of the atmospheric fluorescence yield. The instrument can also be used in the lab to investigate the dependence of the fluorescence yield in air on temperature, pressure and the concentrations of other gases that present in the atmosphere. The results can be used to explore environmental effects on and improve the accuracy of cosmic ray energy measurements for existing ground-based experiments and future space-based experiments.

  5. Fluorescent Applications to Crystallization

    NASA Technical Reports Server (NTRS)

    Pusey, Marc L.; Forsythe, Elizabeth; Achari, Aniruddha

    2006-01-01

    By covalently modifying a subpopulation, less than or equal to 1%, of a macromolecule with a fluorescent probe, the labeled material will add to a growing crystal as a microheterogeneous growth unit. Labeling procedures can be readily incorporated into the final stages of purification, and tests with model proteins have shown that labeling u to 5 percent of the protein molecules does not affect the X-ray data quality obtained . The presence of the trace fluorescent label gives a number of advantages. Since the label is covalently attached to the protein molecules, it "tracks" the protein s response to the crystallization conditions. The covalently attached probe will concentrate in the crystal relative to the solution, and under fluorescent illumination crystals show up as bright objects against a darker background. Non-protein structures, such as salt crystals, do not show up under fluorescent illumination. Crystals have the highest protein concentration and are readily observed against less bright precipitated phases, which under white light illumination may obscure the crystals. Automated image analysis to find crystals should be greatly facilitated, without having to first define crystallization drop boundaries as the protein or protein structures is all that shows up. Fluorescence intensity is a faster search parameter, whether visually or by automated methods, than looking for crystalline features. Preliminary tests, using model proteins, indicates that we can use high fluorescence intensity regions, in the absence of clear crystalline features or "hits", as a means for determining potential lead conditions. A working hypothesis is that more rapid amorphous precipitation kinetics may overwhelm and trap more slowly formed ordered assemblies, which subsequently show up as regions of brighter fluorescence intensity. Experiments are now being carried out to test this approach using a wider range, of proteins. The trace fluorescently labeled crystals will also

  6. Fluorescence endoscopic video system

    NASA Astrophysics Data System (ADS)

    Papayan, G. V.; Kang, Uk

    2006-10-01

    This paper describes a fluorescence endoscopic video system intended for the diagnosis of diseases of the internal organs. The system operates on the basis of two-channel recording of the video fluxes from a fluorescence channel and a reflected-light channel by means of a high-sensitivity monochrome television camera and a color camera, respectively. Examples are given of the application of the device in gastroenterology.

  7. Epi-Fluorescence Microscopy

    PubMed Central

    Webb, Donna J.; Brown, Claire M.

    2012-01-01

    Epi-fluorescence microscopy is available in most life sciences research laboratories, and when optimized can be a central laboratory tool. In this chapter, the epi-fluorescence light path is introduced and the various components are discussed in detail. Recommendations are made for incident lamp light sources, excitation and emission filters, dichroic mirrors, objective lenses, and charge-coupled device (CCD) cameras in order to obtain the most sensitive epi-fluorescence microscope. The even illumination of metal-halide lamps combined with new “hard” coated filters and mirrors, a high resolution monochrome CCD camera, and a high NA objective lens are all recommended for high resolution and high sensitivity fluorescence imaging. Recommendations are also made for multicolor imaging with the use of monochrome cameras, motorized filter turrets, individual filter cubes, and corresponding dyes that are the best choice for sensitive, high resolution multicolor imaging. Images should be collected using Nyquist sampling and should be corrected for background intensity contributions and nonuniform illumination across the field of view. Photostable fluorescent probes and proteins that absorb a lot of light (i.e., high extinction co-efficients) and generate a lot of fluorescence signal (i.e., high quantum yields) are optimal. A neuronal immune-fluorescence labeling protocol is also presented. Finally, in order to maximize the utility of sensitive wide-field microscopes and generate the highest resolution images with high signal-to-noise, advice for combining wide-field epi-fluorescence imaging with restorative image deconvolution is presented. PMID:23026996

  8. A universal rule for organic ligand exchange.

    PubMed

    You, Hongjun; Wang, Wenjin; Yang, Shengchun

    2014-11-12

    Most synthetic routes to high-quality nanocrystals with tunable morphologies predominantly employ long hydro-carbon molecules as ligands, which are detrimental for electronic and catalytic applications. Here, a rule is found that the adsorption energy of an organic ligand is related to its carbon-chain length. Using the density functional theory method, the adsorption energies of some commonly used ligand molecules with different carbon-chain lengths are calculated, including carboxylate, hydroxyl, and amine molecules adsorbed on metal or metal oxide crystal surface. The results indicate that the adsorption energy of the ligand molecule with a long carbon chain is weaker than that of a smaller molecule with same functional group. This rule provides a theoretical support for a new kind of ligand exchange method in which large organic ligand molecules can be exchanged by small molecules with same functional group to improve the catalytic properties. PMID:25335915

  9. Ligand-targeted liposomes for cancer treatment.

    PubMed

    Sapra, Puja; Tyagi, Pradeep; Allen, Theresa M

    2005-10-01

    Selective targeting of ligand-targeted liposomes containing anticancer drugs or therapeutic genes to cell surface receptors expressed on cancer cells is a recognized strategy for improving the therapeutic effectiveness of conventional chemotherapeutics or gene therapeutics. Some recent advances in the field of ligand-targeted liposomes for the treatment of cancer are summarized including: selection criteria for the receptors to be targeted, choice of targeting ligands and choice of encapsulated therapeutics. Targeting of liposomes to solid tumors, versus angiogenic endothelial cells versus vascular targets is discussed. Ligand-targeted liposomes have shown considerable promise in preclinical xenograft models and are poised for clinical development. PMID:16305440

  10. Proton-dependent zinc release from intracellular ligands.

    PubMed

    Kiedrowski, Lech

    2014-07-01

    In cultured cortical and hippocampal neurons when intracellular pH drops from 6.6 to 6.1, yet unclear intracellular stores release micromolar amounts of Zn(2+) into the cytosol. Mitochondria, acidic organelles, and/or intracellular ligands could release this Zn(2+) . Although exposure to the protonophore FCCP precludes reloading of the mitochondria and acidic organelles with Zn(2+) , FCCP failed to compromise the ability of the intracellular stores to repeatedly release Zn(2+) . Therefore, Zn(2+) -releasing stores were not mitochondria or acidic organelles but rather intracellular Zn(2+) ligands. To test which ligands might be involved, the rate of acid-induced Zn(2+) release from complexes with cysteine, glutathione, histidine, aspartate, glutamate, glycine, and carnosine was investigated; [Zn(2+) ] was monitored in vitro using the ratiometric Zn(2+) -sensitive fluorescent probe FuraZin-1. Carnosine failed to chelate Zn(2+) but did chelate Cu(2+) ; the remaining ligands chelated Zn(2+) and upon acidification were releasing it into the medium. However, when pH was decreasing from 6.6 to 6.1, only zinc-cysteine complexes rapidly accelerated the rate of Zn(2+) release. The zinc-cysteine complexes also released Zn(2+) when a histidine-modifying agent, diethylpyrocarbonate, was applied at pH 7.2. Since the cytosolic zinc-cysteine complexes can contain micromolar amounts of Zn(2+) , these complexes may represent the stores responsible for an acid-induced intracellular Zn(2+) release. This study aimed at identifying intracellular stores which release Zn(2+) when pHi drops from 6.6 to 6.1. It was found that these stores are not mitochondria or acidic organelles, but rather intracellular Zn(2+) ligands. When the pH was decreasing from 6.6 to 6.1, only zinc-cysteine complexes showed a rapid acceleration in the rate of Zn(2+) release. Therefore, the stores responsible for an acid-induced intracellular Zn(2+) release in neurons may be the cytosolic zinc-cysteine complexes

  11. A Vinblastine Fluorescent Probe for Pregnane X Receptor in a Time-Resolved Fluorescence Resonance Energy Transfer Assay

    PubMed Central

    Lin, Wenwei; Chen, Taosheng

    2013-01-01

    The pregnane X receptor (PXR) regulates the metabolism and excretion of xenobiotics and endobiotics by regulating the expression of drug-metabolizing enzymes and transporters. The unique structure of PXR allows the binding of many drugs and drug leads to it, possibly causing undesired drug-drug interactions. Therefore, it is crucial to evaluate whether lead compounds bind to PXR. Fluorescence-based assays are preferred because of their sensitivity and non-radioactive nature. One fluorescent PXR probe is currently commercially available; however, because its chemical structure is not publicly disclosed, it is not optimal for studying ligand-PXR interactions. Here we report the characterization of BODIPY FL Vinblastine, generated by labeling vinblastine with the fluorophore 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY FL), as a high-affinity ligand for human PXR with a Kd value of 673 nM. We provide evidence that BODIPY FL Vinblastine is a unique chemical entity different from either vinblastine or the fluorophore 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene in its function as a high-affinity human PXR ligand. We describe a BODIPY FL Vinblastine-based human PXR Time-Resolved Fluorescence Resonance Energy Transfer assay, which was used to successfully test a panel of human PXR ligands. The BODIPY FL Vinblastine–based biochemical assay is suitable for high-throughput screening to evaluate whether lead compounds bind to PXR. PMID:24044991

  12. The Membrane-anchoring Domain of Epidermal Growth Factor Receptor Ligands Dictates Their Ability to Operate in Juxtacrine Mode

    SciTech Connect

    Dong, Jianying; Opresko, Lee; Chrisler, William B.; Orr, Galya; Quesenberry, Ryan D.; Lauffenburger, Douglas A.; Wiley, H S.

    2005-06-01

    All ligands of the epidermal growth factor receptor (EGFR) are synthesized as membrane-anchored precursors. Previous work has suggested that some ligands, such as EGF, must be proteolytically released to be active, whereas others, such as heparin binding EGF-like growth factor (HB-EGF) can function while still anchored to the membrane (i.e., juxtacrine signaling). To explore the structural basis for these differences in ligand activity, we engineered a series of membrane-anchored ligands in which the core, receptor-binding domain of EGF was combined with different domains of both EGF and HB-EGF. We found that ligands having the N-terminal extension of EGF could not bind to the EGFR, even when released from the membrane. Ligands lacking an N-terminal extension, but possessing the membrane-anchoring domain of EGF still required proteolytic release for activity, whereas ligands with the membrane anchoring domain of HB-EGF could elicit full biological activity while still membrane anchored. Ligands containing the HB-EGF membrane anchor, but lacking an N-terminal extension, activated EGFR during their transit through the Golgi apparatus . However, cell-mixing experiments and fluorescence resonance energy transfer (FRET) studies showed that juxtacrine signaling typically occurred in trans at the cell surface, at points of cell-cell contact. Our data suggest that the membrane-anchoring domain of ligands selectively controls their ability to participate in juxtacrine signaling and thus, only a subclass of EGFR ligands can act in a juxtacrine mode.

  13. Quantum dot/glycol chitosan fluorescent nanoconjugates

    NASA Astrophysics Data System (ADS)

    Mansur, Alexandra AP; Mansur, Herman S.

    2015-04-01

    In this study, novel carbohydrate-based nanoconjugates combining chemically modified chitosan with semiconductor quantum dots (QDs) were designed and synthesised via single-step aqueous route at room temperature. Glycol chitosan (G-CHI) was used as the capping ligand aiming to improve the water solubility of the nanoconjugates to produce stable and biocompatible colloidal systems. UV-visible (UV-vis) spectroscopy, photoluminescence (PL) spectroscopy, and Fourier transform infrared (FTIR) spectroscopy were used to characterise the synthesis and the relative stability of biopolymer-capped semiconductor nanocrystals. The results clearly demonstrated that the glycol chitosan derivative was remarkably effective at nucleating and stabilising semiconductor CdS quantum dots in aqueous suspensions under acidic, neutral, and alkaline media with an average size of approximately 2.5 nm and a fluorescent activity in the visible range of the spectra.

  14. Emissive bis-salicylaldiminato Schiff base ligands and their zinc(II) complexes: Synthesis, photophysical properties, mesomorphism and DFT studies

    NASA Astrophysics Data System (ADS)

    Paul, Manoj Kr.; Dilipkumar Singh, Y.; Bedamani Singh, N.; Sarkar, Utpal

    2015-02-01

    Bis-salicylaldiminato Schiff base ligands and their Zn(II) complexes derived from 2,3-Diaminomaleonitrile (DAMN) were synthesized. Their molecular structures, photophysical properties and mesogenic behaviors were investigated. The ligands and their Zn(II) complexes were characterized by using elemental analysis, FT-IR, 1H NMR and molar conductivity measurements. Photophysical properties of ligands and their Zn(II) complexes were investigated in different polar solvents by using UV-visible and fluorescence spectroscopic studies. Ligands emit green light whereas complexes emit orange light upon irradiation with UV-visible light. The liquid crystalline phases of ligands and their Zn(II) complexes were characterized by polarizing optical microscopy and differential scanning calorimetry. The ligand having longer 4-n-octadecyloxy chain (n = 18) displays columnar phase whereas the lower homologues (n = 16, 12) did not show mesophase. The Zn(II) complexes having 4-n-octadecyloxy end chain display smectic B like phase whereas other lower homologues are non mesogenic in nature. The thermal stability of the compounds were studied by using thermo gravimetric analysis. The density functional theory was carried out to obtain the stable molecular conformation, dipole moment, molecular orbitals and polarizability of the ligands and their Zn(II) complexes.

  15. Galactose as Broad Ligand for Multiple Tumor Imaging and Therapy

    PubMed Central

    Ma, Yuxiang; Chen, Haiyan; Su, Shanyuhan; Wang, Tong; Zhang, Congying; Fida, Guissi; Cui, Sisi; Zhao, Juan; Gu, Yueqing

    2015-01-01

    Galactose residues could be specifically recognized by the asialoglycoprotein receptor (ASGPR) which is highly exhibited on liver tissues. However, ASGPR has not been widely investigated on different tumor cell lines except for hepatoma carcinoma cells, which motivates us to investigate the possibility of galactose serving as a board tumor ligand. In this study, a galactose (Gal)-based probe conjugated with fluorescence dye MPA (Gal-MPA) was constructed for the evaluation of tumor affinities/targeted ability on different tumor cell lines. In the vitro cell study, it was indicated that the fluorescence probe Gal-MPA displayed higher cell affinity to tumor cells (HepG2, MCF-7 and A549) than that of the normal liver cells l02. In the vivo dynamic study of Gal-MPA in tumor-bearing mice (HepG2, MCF-7, A549, HCT116, U87, MDA-MB-231 and S180), it was shown that its high tumor targeted ability with the maximal tumor/normal tissue ratio reached up to 6.8. Meanwhile, the fast tumor-targeted ability within 2 hours and long retention on tumor site up to 120 hours were observed. Our results demonstrated that galactose should be a promising broad ligand for multiple tumor imaging and targeted therapy. Subsequently, Gal was covalently conjugated to doxorubicin (DOX) to form prodrug Gal-DOX for tumor targeted therapy. The therapeutic results of Gal-DOX than DOX being better suggested that galactosylated prodrugs might have the prospective potential in tumor targeted therapy. PMID:26078797

  16. A modular phase transfer and ligand exchange protocol for quantum dots.

    PubMed

    Zylstra, Joshua; Amey, Jennifer; Miska, Nathaniel J; Pang, Lisa; Hine, Corey R; Langer, Julia; Doyle, Robert P; Maye, Mathew M

    2011-04-19

    In this paper, we describe a quantum dot (qdot) phase transfer protocol using ligand exchange and the amino acid histidine. The phase transfer from nonpolar solvents to aqueous buffers is homogeneous, and no appreciable precipitation occurs. The molecule histidine was chosen in order to first displace the organic encapsulation and second to provide a weakly chemisorbing intermediate at the qdot ionic interface. This allows the histidine to act as an intermediate shell upon which further direct ligand exchange can occur. Since this intermediate encapsulation is easily displaced by an assortment of different molecules while in aqueous buffers, we refer to this approach as modular. Characterization via FTIR and NMR revealed the extent of ligand exchange, and provides insights into the interfacial binding mechanism. The colloidal stability and photostability of the qdots was probed via UV-vis and steady state fluorescence, which revealed promising quantum yield stability of greater than 1 year. The qdots have hydrodynamic diameters of <12 nm and surface charges dependent upon ligand type and coverage. The modularity of this approach is shown by tailoring the qdot surface charge via sequential ligand exchange using mixed monolayers of carboxylic acid and poly(ethylene glycol)-terminated thiols. PMID:21410215

  17. Thermodynamic fingerprints of ligand binding to human telomeric G-quadruplexes.

    PubMed

    Bončina, Matjaž; Podlipnik, Črtomir; Piantanida, Ivo; Eilmes, Julita; Teulade-Fichou, Marie-Paule; Vesnaver, Gorazd; Lah, Jurij

    2015-12-01

    Thermodynamic studies of ligand binding to human telomere (ht) DNA quadruplexes, as a rule, neglect the involvement of various ht-DNA conformations in the binding process. Therefore, the thermodynamic driving forces and the mechanisms of ht-DNA G-quadruplex-ligand recognition remain poorly understood. In this work we characterize thermodynamically and structurally binding of netropsin (Net), dibenzotetraaza[14]annulene derivatives (DP77, DP78), cationic porphyrin (TMPyP4) and two bisquinolinium ligands (Phen-DC3, 360A-Br) to the ht-DNA fragment (Tel22) AGGG(TTAGGG)3 using isothermal titration calorimetry, CD and fluorescence spectroscopy, gel electrophoresis and molecular modeling. By global thermodynamic analysis of experimental data we show that the driving forces characterized by contributions of specific interactions, changes in solvation and conformation differ significantly for binding of ligands with low quadruplex selectivity over duplexes (Net, DP77, DP78, TMPyP4; KTel22 ≈ ligands (Phen-DC3, 360A-Br; KTel22 > KdsDNA). These contributions are in accordance with the observed structural features (changes) and suggest that upon binding Net, DP77, DP78 and TMPyP4 select hybrid-1 and/or hybrid-2 conformation while Phen-DC3 and 360A-Br induce the transition of hybrid-1 and hybrid-2 to the structure with characteristics of antiparallel or hybrid-3 type conformation. PMID:26546516

  18. Nuclear receptor ligand-binding domains: reduction of helix H12 dynamics to favour crystallization

    SciTech Connect

    Nahoum, Virginie; Lipski, Alexandra; Quillard, Fabien; Guichou, Jean-François; Boublik, Yvan; Pérez, Efrèn; Germain, Pierre; Lera, Angel R. de; Bourguet, William

    2008-07-01

    Attempts have been made to crystallize the ligand-binding domain of the human retinoid X receptor in complex with a variety of newly synthesized ligands. An inverse correlation was observed between the ‘crystallizability’ and the structural dynamics of the various receptor–ligand complexes. Crystallization trials of the human retinoid X receptor α ligand-binding domain (RXRα LBD) in complex with various ligands have been carried out. Using fluorescence anisotropy, it has been found that when compared with agonists these small-molecule effectors enhance the dynamics of the RXRα LBD C-terminal helix H12. In some cases, the mobility of this helix could be dramatically reduced by the addition of a 13-residue co-activator fragment (CoA). In keeping with these observations, crystals have been obtained of the corresponding ternary RXRα LBD–ligand–CoA complexes. In contrast, attempts to crystallize complexes with a highly mobile H12 remained unsuccessful. These experimental observations substantiate the previously recognized role of co-regulator fragments in facilitating the crystallization of nuclear receptor LBDs.

  19. Small antibody-like proteins with prescribed ligand specificities derived from the lipocalin fold

    PubMed Central

    Beste, Gerald; Schmidt, Frank S.; Stibora, Thomas; Skerra, Arne

    1999-01-01

    We demonstrate that the ligand pocket of a lipocalin from Pieris brassicae, the bilin-binding protein (BBP), can be reshaped by combinatorial protein design such that it recognizes fluorescein, an established immunological hapten. For this purpose 16 residues at the center of the binding site, which is formed by four loops on top of an eight-stranded β-barrel, were subjected to random mutagenesis. Fluorescein-binding BBP variants were then selected from the mutant library by bacterial phage display. Three variants were identified that complex fluorescein with high affinity, exhibiting dissociation constants as low as 35.2 nM. Notably, one of these variants effects almost complete quenching of the ligand fluorescence, similarly as an anti-fluorescein antibody. Detailed ligand-binding studies and site-directed mutagenesis experiments indicated (i) that the molecular recognition of fluorescein is specific and (ii) that charged residues at the center of the pocket are responsible for tight complex formation. Sequence comparison of the BBP variants directed against fluorescein with the wild-type protein and with further variants that were selected against several other ligands revealed that all of the randomized amino acid positions are variable. Hence, a lipocalin can be used for generating molecular pockets with a diversity of shapes. We term this class of engineered proteins “anticalins.” Their one-domain scaffold makes them a promising alternative to antibodies to create a stable receptor protein for a ligand of choice. PMID:10051566

  20. Towards a Dithiocarbamate Ligand for CdS Nanoparticle-based Photocatalysis

    NASA Astrophysics Data System (ADS)

    O'Hara, Andrew; Lacroix, Andrew D.; Pantelides, Sokrates T.; MacDonald, Janet E.

    Photocatalysis of water into H2 and O2 presents a clean, renewable route for energy storage and production. Traditionally, most semiconducting nanoparticle research on photocatalysis has focused on the ability to reduce chemical systems using the photoexcited electron. Here we employ a combination of theory and experiments to develop a possible route towards the oxidation of chemical systems via the hole from photoexcitation using an asymmetric bipyridine ligand with conjugated dithiocarbamate ligand bound to the surface of cadmium sulfide nanorods. In particular, we use density functional theory to calculate the electronic levels and optical absorption of the designer ligand, free from the cadmium sulfide surface as well as attached to the surface, with and without the copper center. These calculations are compared with experimental UV/VIS absorption and fluorescence spectroscopy measurements to understand the role of copper chelation. Furthermore, theoretical comparisons are made with a related ligand known to oxidize water under an applied potential bias. Finally, we discuss whether we expect photocatalysis from the ligand and possible improvements to its design.

  1. Thermodynamic fingerprints of ligand binding to human telomeric G-quadruplexes

    PubMed Central

    Bončina, Matjaž; Podlipnik, Črtomir; Piantanida, Ivo; Eilmes, Julita; Teulade-Fichou, Marie-Paule; Vesnaver, Gorazd; Lah, Jurij

    2015-01-01

    Thermodynamic studies of ligand binding to human telomere (ht) DNA quadruplexes, as a rule, neglect the involvement of various ht-DNA conformations in the binding process. Therefore, the thermodynamic driving forces and the mechanisms of ht-DNA G-quadruplex-ligand recognition remain poorly understood. In this work we characterize thermodynamically and structurally binding of netropsin (Net), dibenzotetraaza[14]annulene derivatives (DP77, DP78), cationic porphyrin (TMPyP4) and two bisquinolinium ligands (Phen-DC3, 360A-Br) to the ht-DNA fragment (Tel22) AGGG(TTAGGG)3 using isothermal titration calorimetry, CD and fluorescence spectroscopy, gel electrophoresis and molecular modeling. By global thermodynamic analysis of experimental data we show that the driving forces characterized by contributions of specific interactions, changes in solvation and conformation differ significantly for binding of ligands with low quadruplex selectivity over duplexes (Net, DP77, DP78, TMPyP4; KTel22 ≈ ligands (Phen-DC3, 360A-Br; KTel22 > KdsDNA). These contributions are in accordance with the observed structural features (changes) and suggest that upon binding Net, DP77, DP78 and TMPyP4 select hybrid-1 and/or hybrid-2 conformation while Phen-DC3 and 360A-Br induce the transition of hybrid-1 and hybrid-2 to the structure with characteristics of antiparallel or hybrid-3 type conformation. PMID:26546516

  2. Recent development of dihydrolipoic acid appended ligands for robust and biocompatible quantum dots

    NASA Astrophysics Data System (ADS)

    Susumu, Kimihiro; Oh, Eunkeu; Stewart, Michael H.; Delehanty, James B.; Gemmill, Kelly B.; Jain, Vaibhav; Huston, Alan L.; Medintz, Igor L.

    2013-02-01

    Biocompatible nanoparticles have recently attracted significant attention due to increasing interest in their use for biological sensing, cellular labeling and in vivo imaging. Semiconductor quantum dots (QDs) with good colloidal stability as well as small hydrodynamic sizes are particularly useful within these applications. We have developed a series of dihydrolipoic acid (DHLA) based surface ligands to enhance the colloidal stability and biocompatibility of water soluble QDs. Modification of DHLA with poly(ethylene glycol) derivatives provided the QDs with extended colloidal stability over a wide pH range and under high salt concentrations, which contrasts with the limited colloidal stability provided by DHLA alone. Functionalization of the PEG termini enabled one to have easy access to the QD surface and construct a variety of stable QD-biomolecules conjugates. A series of DHLA-based compact ligands with zwitterionic character has also been explored to develop compact sized QDs without sacrificing the colloidal stability. Despite their smaller sizes than the PEG analogs, the QDs coated with the zwitterionic ligands still have excellent colloidal stability and minimize nonspecific interactions in biological environments. Recent studies of thiol-based multidentate ligands and ligand exchange methods further improved the colloidal stability and fluorescence quantum yields.

  3. A reversible dual-response fluorescence switch for the detection of multiple analytes.

    PubMed

    Geng, Junlong; Liu, Ping; Liu, Bianhua; Guan, Guijian; Zhang, Zhongping; Han, Ming-Yong

    2010-03-22

    This paper reports a reversible dual fluorescence switch for the detection of a proton target and 2,4,6-trinitrotoluene (TNT) with opposite-response results, based on fluorophore derivatization of silica nanoparticles. Fluorescent silica nanoparticles were synthesized through modification of the surface with a nitrobenzoxadiazole (NBD) fluorophore and an organic amine to form a hybrid monolayer of fluorophores and amino ligands; the resultant nanoparticles showed different fluorescence responses to the proton target and TNT. Protonation of the amino ligands leads to fluorescence enhancement due to inhibition of photoinduced electron transfer (PET) between the amine and fluorophore. By contrast, addition of TNT results in fluorescence quenching because a fluorescence resonance energy transfer (FRET) happens between the NBD fluorophore and the formed TNT-amine complex. The fluorescence signal is reversible through washing with the proper solvents and the nanoparticles can be reused after centrifugal separation. Furthermore, these nanoparticles were assembled into chips on an etched silicon wafer for the detection of TNT and the proton target. The assembled chip can be used as a convenient indicator of herbicide (2,4-dichlorophenoxyacetic acid) and TNT residues with the use of only 10 microL of sample. The simple NBD-grafted silica nanoparticles reported here show a reversible signal and good assembly flexibility; thus, they can be applied in multianalyte detection. PMID:20151433

  4. Fluorescent image tracking velocimeter

    DOEpatents

    Shaffer, Franklin D.

    1994-01-01

    A multiple-exposure fluorescent image tracking velocimeter (FITV) detects and measures the motion (trajectory, direction and velocity) of small particles close to light scattering surfaces. The small particles may follow the motion of a carrier medium such as a liquid, gas or multi-phase mixture, allowing the motion of the carrier medium to be observed, measured and recorded. The main components of the FITV include: (1) fluorescent particles; (2) a pulsed fluorescent excitation laser source; (3) an imaging camera; and (4) an image analyzer. FITV uses fluorescing particles excited by visible laser light to enhance particle image detectability near light scattering surfaces. The excitation laser light is filtered out before reaching the imaging camera allowing the fluoresced wavelengths emitted by the particles to be detected and recorded by the camera. FITV employs multiple exposures of a single camera image by pulsing the excitation laser light for producing a series of images of each particle along its trajectory. The time-lapsed image may be used to determine trajectory and velocity and the exposures may be coded to derive directional information.

  5. Oxytocin receptor ligands induce changes in cytoskeleton in neuroblastoma cells.

    PubMed

    Bakos, Jan; Strbak, Vladimir; Paulikova, Helena; Krajnakova, Lucia; Lestanova, Zuzana; Bacova, Zuzana

    2013-07-01

    Aim of the present study was to evaluate effects of ligands of oxytocin receptors on gene expression of neurofilament proteins (nestin and microtubule-associated protein 2 (MAP2)) associated with neuronal differentiation and growth factors (brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF)) related to neuronal growth. Fluorescent staining of F-actin was used to observe morphology of cells. Co-treatment with oxytocin and oxytocin receptor antagonist--atosiban--resulted in significant increase of MAP2 gene expression in SK-N-SH cells. There was no effect of oxytocin on gene expression of growth factors BDNF and NGF. Surprisingly, oxytocin with atosiban significantly increased mRNA levels for both BDNF and NGF. Gene expression of vasopressin receptor (V1aR) significantly decreased in response to vasopressin. Atosiban decreased mRNA levels for oxytocin receptor (OXTR) and V1aR. Oxytocin significantly decreased OXTR and nestin mRNA levels and increased mRNA levels for BDNF and NGF in U-87 MG cells. The densest recruitment of F-actin filaments was observed in apical parts of filopodia in SK-N-SH cells incubated in oxytocin presence. Present data demonstrate complex role of ligands of oxytocin receptors in regulation of gene expression of intermediate filaments and thus, oxytocin might be considered as a growth factor in neuronal type of cells. PMID:23335033

  6. Silver Nanoclusters with Specific Ion Recognition Modulated by Ligand Passivation toward Fluorimetric and Colorimetric Copper Analysis and Biological Imaging

    PubMed Central

    Sun, Zongzhao; Li, Shuying; Jiang, Yao; Qiao, Yuchun; Zhang, Liyan; Xu, Lulu; Liu, Jinghui; Qi, Wei; Wang, Hua

    2016-01-01

    Silver nanoclusters were synthesized and passivated by glutathione (GSH) ligand, with high aqueous stability and powerful red fluorescence and UV-vis yellow colour. Importantly, the specific recognition of the AgNCs was modulated from Hg2+ ions to Cu2+ ions upon the GSH passivation, of which the unique GSH-Cu2+ chelating reaction could conduct the fluorescence quenching of AgNCs. Strong UV-vis absorbance of GSH-passivated AgNCs could also be realized depending on the Cu2+ levels. Moreover, the Cu2+-induced loss of fluorescence and UV-vis absorbance of GSH-passivated AgNCs could be well restored by using stronger Cu2+ chelating agent. A simultaneous and reversible fluorimetric and colorimetric sensing method was thereby developed for probing Cu2+ ions in blood with high sensitivity and selectivity. Subsequently, the fluorescence-trackable imaging for live tissues and cells was demonstrated towards the analysis Cu2+ ions using GSH-passivated AgNCs as the fluorescent probes. This study indicates that the use of functional ligands like GSH could not only modulate the specific ion recognition of AgNCs, but also endow them the high aqueous stability and powerful red fluorescence towards the wide applications for ion sensing and biological imaging in the complicated media like blood. PMID:26847593

  7. Silver Nanoclusters with Specific Ion Recognition Modulated by Ligand Passivation toward Fluorimetric and Colorimetric Copper Analysis and Biological Imaging

    NASA Astrophysics Data System (ADS)

    Sun, Zongzhao; Li, Shuying; Jiang, Yao; Qiao, Yuchun; Zhang, Liyan; Xu, Lulu; Liu, Jinghui; Qi, Wei; Wang, Hua

    2016-02-01

    Silver nanoclusters were synthesized and passivated by glutathione (GSH) ligand, with high aqueous stability and powerful red fluorescence and UV-vis yellow colour. Importantly, the specific recognition of the AgNCs was modulated from Hg2+ ions to Cu2+ ions upon the GSH passivation, of which the unique GSH-Cu2+ chelating reaction could conduct the fluorescence quenching of AgNCs. Strong UV-vis absorbance of GSH-passivated AgNCs could also be realized depending on the Cu2+ levels. Moreover, the Cu2+-induced loss of fluorescence and UV-vis absorbance of GSH-passivated AgNCs could be well restored by using stronger Cu2+ chelating agent. A simultaneous and reversible fluorimetric and colorimetric sensing method was thereby developed for probing Cu2+ ions in blood with high sensitivity and selectivity. Subsequently, the fluorescence-trackable imaging for live tissues and cells was demonstrated towards the analysis Cu2+ ions using GSH-passivated AgNCs as the fluorescent probes. This study indicates that the use of functional ligands like GSH could not only modulate the specific ion recognition of AgNCs, but also endow them the high aqueous stability and powerful red fluorescence towards the wide applications for ion sensing and biological imaging in the complicated media like blood.

  8. Screening and Optimization of Ligand Conjugates for Lysosomal Targeting

    PubMed Central

    Meerovich, Igor; Koshkaryev, Alexander; Thekkedath, Ritesh; Torchilin, Vladimir P.

    2011-01-01

    The use of lysosome-targeted liposomes may significantly improve the delivery of therapeutic enzymes and chaperones into lysosomes for the treatment of lysosomal storage disorders. The aim of this research was to synthesize new potentially lysosomotropic ligands on a base of Neutral Red and rhodamine B and to study their ability to enhance specific lysosomal delivery of surface-modified liposomes loaded with a model compound, fluorescein isothiocyanate-dextran (FD). The delivery of these liposomes and their content to lysosomes in HeLa cells was investigated by confocal immunofluorescent microscopy, subcellular fractionation and flow cytometry. Confocal microscopy demonstrated that liposomes modified with derivatives of rhodamine B provide good rate of co-localization well the specific lysosomal markers. The comparison of fluorescence of FD in lysosomes isolated by subcellular fractionation also showed that the efficiency of lysosomal delivery of liposomal load by liposomes modified with some of synthesized ligands was significantly higher compared with plain liposomes. These results were additionally confirmed by the flow cytometry of the intact cells treated with liposomes loaded with with 5-dodecanoylaminofluorescein di-β-D-galactopyranoside, a specific substrate for the intralysosomal β-galactosidase, using a number of cell lines, including macrophages with induced phenotype of lysosomal enzyme deficiency; two of the synthesized ligands – rhodamine B DSPE-PEG2k-amide and 6-(3-(DSPE-PEG2k)-thioureido) rhodamine B – demonstrated enhanced lysosomal delivery, in some cases, higher than that for commercially available rhodamine B octadecyl ester, with the best results (the enhancement of the lysosomal delivery up to 75% greater in comparison to plain liposomes) shown for the cells with induced lysosomal enzyme deficiency phenotype. Use of liposomes modified with rhodamine B derivatives may be advantageous for the development of drug delivery systems for the

  9. Development of a competitive fluorescence-based synaptosome binding assay for brevetoxins

    PubMed Central

    McCall, Jennifer R.; Jacocks, Henry M.; Baden, Daniel G.; Bourdelais, Andrea J.

    2012-01-01

    Brevetoxins are a family of ladder-frame polyether toxins produced during blooms of the marine dinoflagellate Karenia brevis. Inhalation of brevetoxins aerosolized by wind and wave action can lead to asthma-like symptoms in beach goers. Consumption of either shellfish or finfish exposed to K. brevis blooms can lead to the development of neurotoxic shellfish poisoning. The toxic effects of brevetoxins are due to activation of voltage-sensitive sodium channels (VSSCs) in cell membranes. Binding of brevetoxin analogs and competitors to site 5 on these channels has historically been measured using a radioligand competition assay that is fraught with difficulty, including slow analysis time, production of radioactive waste, and cumbersome and expensive methods associated with the generation of radioactive labeled ligands. In this study, we describe the development of a novel fluorescent synaptosome binding assay for the brevetoxin receptor. BODIPY®-conjugated to PbTx-2 was used as the labeled ligand. The BODIPY®-PbTx-2 conjugate was found to displace [3H]-PbTx-3 from its binding site on VSSCs on rat brain synaptosomes with an equilibrium inhibition constant of 0.11 nM. We have shown that brevetoxin A and B analogs are all able to compete for binding with the fluorescent ligand. Most importantly, this assay was validated against the current site 5 receptor binding assay standard, the radioligand receptor assay for the brevetoxin receptor using [3H]-PbTx-3 as the labeled ligand. The fluorescence based assay yielded equilibrium inhibition constants comparable to the radioligand assay for all brevetoxin analogs. The fluorescence based assay was quicker, far less expensive, and did not generate radioactive waste or need radioactive facilities. As such, this fluorescence-based assay can be used to replace the current radioligand assay for site 5 on voltage-sensitive sodium channels and will be a vital tool for future experiments examining the binding affinity of various

  10. The important role of surface ligand on CdSe/CdS core/shell nanocrystals in affecting the efficiency of H2 photogeneration from water

    NASA Astrophysics Data System (ADS)

    Wang, Ping; Zhang, Jie; He, Haili; Xu, Xiaolong; Jin, Yongdong

    2015-03-01

    The use of colloidal semiconductor nanocrystals (NCs), especially those with a core/shell structure, for photocatalytic hydrogen (H2) production from water is currently one of the hottest research fields. Although the ligand on the semiconductor NC surface is crucial to the optical and optoelectronic properties of the NC, the study of the ligand effect on the photocatalytic activity of H2 generation is rarely reported. Herein, we employ nearly monodispersed CdSe/CdS core/shell NCs as a model photocatalytic system, and three kinds of ligands with different numbers of functional thiol groups (i.e., poly(acrylic acid), 3-mercaptopropionic acid and 2,3-dimercaptosuccinic acid) are selected as the ligands to investigate the effect of ligand on the efficiency of H2 photogeneration. The results show that the H2 photogeneration efficiency is highly dependent on the surface ligand of the NCs, and it increases with the increase of the number of the functional thiol groups in the ligand, and correspondingly, the photoluminescence intensity and average fluorescence lifetime, which are measured by steady state and time-resolved fluorescence measurements, are decreased. The surface trap-related charge separation efficiency, which is mediated by surface coating with different ligands, is supposed to cause the distinct ligand-dependent performance in the H2 evolution.The use of colloidal semiconductor nanocrystals (NCs), especially those with a core/shell structure, for photocatalytic hydrogen (H2) production from water is currently one of the hottest research fields. Although the ligand on the semiconductor NC surface is crucial to the optical and optoelectronic properties of the NC, the study of the ligand effect on the photocatalytic activity of H2 generation is rarely reported. Herein, we employ nearly monodispersed CdSe/CdS core/shell NCs as a model photocatalytic system, and three kinds of ligands with different numbers of functional thiol groups (i.e., poly(acrylic acid), 3

  11. Allosterism at muscarinic receptors: ligands and mechanisms.

    PubMed

    Birdsall, N J M; Lazareno, S

    2005-06-01

    The evaluation of allosteric ligands at muscarinic receptors is discussed in terms of the ability of the experimental data to be interpreted by the allosteric ternary complex model. The compilation of useful SAR information of allosteric ligands is not simple, especially for muscarinic receptors, where there are multiple allosteric sites and complex interactions. PMID:15974931

  12. Autocrine signal transmission with extracellular ligand degradation

    NASA Astrophysics Data System (ADS)

    Muratov, C B; Posta, F; Shvartsman, S Y

    2009-03-01

    Traveling waves of cell signaling in epithelial layers orchestrate a number of important processes in developing and adult tissues. These waves can be mediated by positive feedback autocrine loops, a mode of cell signaling where binding of a diffusible extracellular ligand to a cell surface receptor can lead to further ligand release. We formulate and analyze a biophysical model that accounts for ligand-induced ligand release, extracellular ligand diffusion and ligand-receptor interaction. We focus on the case when the main mode for ligand degradation is extracellular and analyze the problem with the sharp threshold positive feedback nonlinearity. We derive expressions that link the speed of propagation and other characteristics of traveling waves to the parameters of the biophysical processes, such as diffusion rates, receptor expression level, etc. Analyzing the derived expressions we found that traveling waves in such systems can exhibit a number of unusual properties, e.g. non-monotonic dependence of the speed of propagation on ligand diffusivity. Our results for the fully developed traveling fronts can be used to analyze wave initiation from localized perturbations, a scenario that frequently arises in the in vitro models of epithelial wound healing, and guide future modeling studies of cell communication in epithelial layers.

  13. Use of induced fluorescence measurements to assess aluminum-organic interactions in acidified lakes

    NASA Technical Reports Server (NTRS)

    Vodacek, A.; Philpot, W. D.

    1985-01-01

    The application of laser fluorosensing to the tracing of metals in acid lakes is proposed. The effects of the metals on the dissolving organic carbon (DOC) fluorescence is studied using laboratory mixed water samples and natural water samples from Hamilton and Big Moose Lakes in New York. The operation of the laser fluorosensing system employed in the experiment is described. The DOC fluorescence was quenched by Al, Cu, and Fe, and the relation between pH and the quenching rate is examined. The humic substances fluorescence spectra are analyzed to estimate the concentrations of DOC in water and the relative concentration of Al. The interference problems caused by chemical competition between metal ions and ligands, and changes in the background DOC fluorescence are discussed. It is noted that an airborne laser fluorescence is useful for detecting elevated concentrations of metals.

  14. Fiberized fluorescent dye microtubes

    NASA Astrophysics Data System (ADS)

    Vladev, Veselin; Eftimov, Tinko

    2013-03-01

    In the present work we study the effect of the length of fluorescent dye-filled micro-capillaries on the fluorescence spectra. Two types of micro-capillaries have been studied: a 100 μm inner diameter fused silica capillary with a transparent coating and one of the holes of a fiber optic glass ferrule with 125 μm inner diameter. The tubes were filled with solutions of Rhodamine 6G dissolved in ethanol and then in glycerin. Experimental data show that the maximum fluorescence and the largest spectral widths are observed for a sample length of about 0.25 mm for the used concentration. This results show that miniature tunable fiberized dye lasers can be developed using available standard micro-and fibre-optic components.

  15. Optically trapped fluorescent nanodiamonds

    NASA Astrophysics Data System (ADS)

    Horowitz, Viva R.; Alemán, Benjamin J.; Christle, David; Cleland, Andrew N.; Awschalom, David D.

    2012-02-01

    The electronic spin state of the nitrogen-vacancy (NV) center in diamond has gained considerable interest because it can be optically initialized, coherently manipulated, and optically read out at room temperature. In addition, nanoparticle diamonds containing NV centers can be integrated with biological and microfluidic systems. We have constructed and characterized an optical tweezers apparatus to trap fluorescent nanodiamonds in a fluid and measure their fluorescence. Particles are held and moved in three dimensions using an infrared trapping laser. Fluorescent detection of these optically trapped nanodiamonds enables us to observe nanoparticle dynamics and to measure electron spin resonance of NV centers. We will discuss applications using the electron spin resonance of trapped NV centers in nanodiamonds for magnetic field imaging in fluidic environments.

  16. Pre-Assembly of Near-Infrared Fluorescent Multivalent Molecular Probes for Biological Imaging.

    PubMed

    Peck, Evan M; Battles, Paul M; Rice, Douglas R; Roland, Felicia M; Norquest, Kathryn A; Smith, Bradley D

    2016-05-18

    A programmable pre-assembly method is described and shown to produce near-infrared fluorescent molecular probes with tunable multivalent binding properties. The modular assembly process threads one or two copies of a tetralactam macrocycle onto a fluorescent PEGylated squaraine scaffold containing a complementary number of docking stations. Appended to the macrocycle periphery are multiple copies of a ligand that is known to target a biomarker. The structure and high purity of each threaded complex was determined by independent spectrometric methods and also by gel electrophoresis. Especially helpful were diagnostic red-shift and energy transfer features in the absorption and fluorescence spectra. The threaded complexes were found to be effective multivalent molecular probes for fluorescence microscopy and in vivo fluorescence imaging of living subjects. Two multivalent probes were prepared and tested for targeting of bone in mice. A pre-assembled probe with 12 bone-targeting iminodiacetate ligands produced more bone accumulation than an analogous pre-assembled probe with six iminodiacetate ligands. Notably, there was no loss in probe fluorescence at the bone target site after 24 h in the living animal, indicating that the pre-assembled fluorescent probe maintained very high mechanical and chemical stability on the skeletal surface. The study shows how this versatile pre-assembly method can be used in a parallel combinatorial manner to produce libraries of near-infrared fluorescent multivalent molecular probes for different types of imaging and diagnostic applications, with incremental structural changes in the number of targeting groups, linker lengths, linker flexibility, and degree of PEGylation. PMID:27088305

  17. Fluorescent derivative of cysteine-10 reveals thyroxine-dependent conformational modifications in human serum prealbumin.

    PubMed

    González, G

    1989-05-15

    Fluorescence studies on the N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine-labeled cysteine-10 residue of human prealbumin were carried out to detect conformational changes induced by the binding of the ligand thyroxine to the two structurally identical binding sites. A red shift of the spectrum was observed and the total change was confined to the first ligand. This was interpreted as resulting from a conformational change which increases the exposure of the fluorescent probe moiety. Thyroxine also alters the effect of the collisional quencher, acrylamide, confirming the greater exposure of the probe. This modification in structure is associated with changes in relaxation time which indicate that when thyroxine is bound there is an increase in the rotational freedom of the segment or domain of prealbumin which contains the fluorescent probe. PMID:2712572

  18. Distribution and Dynamics of Rat Basophilic Leukemia Immunoglobulin E Receptors (FcɛRI) on Planar Ligand-Presenting Surfaces

    PubMed Central

    Spendier, Kathrin; Carroll-Portillo, Amanda; Lidke, Keith A.; Wilson, Bridget S.; Timlin, Jerilyn A.; Thomas, James L.

    2010-01-01

    Abstract There is considerable interest in the signaling mechanisms of immunoreceptors, especially when triggered with membrane-bound ligands. We have quantified the spatiotemporal dynamics of the redistribution of immunoglobulin E-loaded receptors (IgE-FcɛRI) on rat basophilic leukemia-2H3 mast cells in contact with fluid and gel-phase membranes displaying ligands for immunoglobulin E, using total internal reflection fluorescence microscopy. To clearly separate the kinetics of receptor redistribution from cell spreading, and to precisely define the initial contact time (±50 ms), micropipette cell manipulation was used to bring individual cells into contact with surfaces. On ligand-free surfaces, there are micron-scale heterogeneities in fluorescence that likely reflect regions of the cell that are more closely apposed to the substrate. When ligands are present, receptor clusters form with this same size scale. The initial rate of accumulation of receptors into the clusters is consistent with diffusion-limited trapping with D ∼10−1μm2/s. These results support the hypothesis that clusters form by diffusion to cell-surface contact regions. Over longer timescales (>10 s), individual clusters moved with both diffusive and directed motion components. The dynamics of the cluster motion is similar to the dynamics of membrane fluctuations of cells on ligand-free fluid membranes. Thus, the same cellular machinery may be responsible for both processes. PMID:20643056

  19. Smartphone fluorescence spectroscopy.

    PubMed

    Yu, Hojeong; Tan, Yafang; Cunningham, Brian T

    2014-09-01

    We demonstrate the first use of smartphone spectrophotometry for readout of fluorescence-based biological assays. We evaluated the smartphone fluorimeter in the context of a fluorescent molecular beacon (MB) assay for detection of specific nucleic acid sequences in a liquid test sample and compared performance against a conventional laboratory fluorimeter. The capability of distinguishing a one-point mismatch is also demonstrated by detecting single-base mutation in target nucleic acids. Our approach offers a route toward portable biomolecular assays for viral/bacterial pathogens, disease biomarkers, and toxins. PMID:25098859

  20. Fast fluorescence holographic microscopy

    PubMed Central

    Qin, Wan; Yang, Xiaoqi; Li, Yingying; Peng, Xiang; Qu, Xinghua; Yao, Hai; Gao, Bruce Z.

    2015-01-01

    FINCHSCOPE is a new technology of fluorescence holographic microscopy. It has been successfully applied to recording high-resolution three-dimensional fluorescence images of biological specimens without the need for scanning. In this study, we revealed and analyzed an intrinsic phenomenon, called ghost lens effect, on spatial light modulator which is the core element enabling the incoherent correlation in the FINCHSCOPE. The ghost lens effect can degrade the imaging quality by introducing multiple spherical waves with different focal lengths into the correlation and thus increasing the noise in the recorded holograms. PMID:25767693

  1. Fluorescence activated cell sorting.

    NASA Technical Reports Server (NTRS)

    Bonner, W. A.; Hulett, H. R.; Sweet, R. G.; Herzenberg, L. A.

    1972-01-01

    An instrument has been developed for sorting biological cells. The cells are rendered differentially fluorescent and incorporated into a small liquid stream illuminated by a laser beam. The cells pass sequentially through the beam, and fluorescent light from the cells gives rise to electrical signals. The stream is broken into a series of uniform size drops downstream of the laser. The cell signals are used to give appropriate electrostatic charges to drops containing the cells. The drops then pass between two charged plates and are deflected to appropriate containers. The system has proved capable of providing fractions containing large numbers of viable cells highly enriched in a particular functional type.

  2. Smartphone fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Yu, Hojoeng; Tan, Yafang; Cunningham, Brian T.

    2014-03-01

    We demonstrate the first use of smartphone spectrophotometry for readout of fluorescence-based biological assays. We evaluated the smartphone fluorimeter in the context of a fluorescent molecular beacon (MB) assay for detection of a specific nucleic acid sequences in a liquid test sample. The capability of distinguishing a one-point mismatch is also demonstrated by detecting single-base mutation in target nucleic acids. Our approach offers a route towards portable biomolecular assays for viral/bacterial pathogens, disease biomarkers, and toxins.

  3. Nanosecond fluorescence spectroscopy

    SciTech Connect

    Leskovar, B.

    1985-03-01

    This article is a summary of a short course lecture given in conjunction with the 1984 Nuclear Science Symposium. Measuring systems for nanosecond fluorescence spectroscopy using single-photon counting techniques are presented. These involve systems based on relaxation-type spark gap light pulser and synchronously pumped mode-locked dye lasers. Furthermore, typical characteristics and optimization of operating conditions of the critical components responsible for the system time resolution are discussed. A short comparison of the most important deconvolution methods for numerical analysis of experimental data is given particularly with respect to the signal-to-noise ratio of the fluorescence signal. 22 refs., 8 figs.

  4. Synthesis, characterization, DNA binding properties, fluorescence studies and antioxidant activity of transition metal complexes with hesperetin-2-hydroxy benzoyl hydrazone.

    PubMed

    Li, Yong; Yang, Zheng-Yin; Wang, Ming-Fang

    2010-07-01

    A novel Schiff-base ligand (H(5)L), hesperetin-2-hydroxy benzoyl hydrazone, and its copper (II), zinc (II) and nickel (II) complexes (M.H(3)L) [M(II) = Cu, Zn, Ni], have been synthesized and characterized. The ligand and Zn (II) complex exhibit green and blue fluorescence under UV light and the fluorescent properties of the ligand and Zn (II) complex in solid state and different solutions were investigated. In addition, DNA binding properties of the ligand and its metal complexes have been investigated by electronic absorption spectroscopy, fluorescence spectra, ethidium bromide displacement experiments, iodide quenching experiments, salt effect and viscosity measurements. Results suggest that all the compounds bind to DNA via an intercalation binding mode. Furthermore, the antioxidant activity of the ligand and its metal complexes was determined by superoxide and hydroxyl radical scavenging methods in vitro. The metal complexes were found to possess potent antioxidant activity and be better than the free ligand alone and some standard antioxidants like vitamin C and mannitol. PMID:20352308

  5. Tryptophan and tyrosine to terbium fluorescence resonance energy transfer as a method to 'map' aromatic residues and monitor docking

    SciTech Connect

    Allen, John E.; McLendon, George L. . E-mail: george.mclendon@duke.edu

    2006-11-03

    Fluorescent lanthanide ions, with large Stokes shifts and narrow emission bands, are excellent tools for the development of FRET-based assays. In this work, a terbium ion is tethered to a peptide which binds to the BIR3 domain of XIAP, an anti-apoptotic protein. Excitation of tryptophan and tyrosine residues in the BIR3 domain causes the peptide bound terbium ion to fluoresce relative to its distance from these aromatic residues. By developing ligands with terbium ions tethered at different residues, the relative terbium emission can be used to 'map' the aromatic residues within the ligand binding pocket.

  6. Complementation and Reconstitution of Fluorescence from Circularly Permuted and Truncated Green Fluorescent Protein†

    PubMed Central

    Huang, Yao-ming; Bystroff, Christopher

    2015-01-01

    Green fluorescent protein (GFP) has been used as a proof of concept for a novel “leave-one-out” biosensor design in which a protein that has a segment omitted from the middle of the sequence by circular permutation and truncation binds the missing peptide and reconstitutes its function. Three variants of GFP have been synthesized that are each missing one of the 11 β-strands from its β-barrel structure, and in two of the variants, adding the omitted peptide sequence in trans reconstitutes fluorescence. Detailed biochemical analysis indicates that GFP with β-strand 7 “left out” (t7SPm) exists in a partially unfolded state. The apo form t7SPm binds the free β-strand 7 peptide with a dissociation constant of ~0.5 µM and folds into the native state of GFP, resulting in fluorescence recovery. Folding of t7SPm, both with and without the peptide ligand, is at least a three-state process and has a rate comparable to that of the full-length and unpermuted GFP. The conserved kinetic properties strongly suggest that the rate-limiting steps in the folding pathway have not been altered by circular permutation and truncation in t7SPm. This study shows that structural and functional reconstitution of GFP can occur with a segment omitted from the middle of the chain, and that the unbound form is in a partially unfolded state. PMID:19140681

  7. Fluorescent chelates for monitoring metal binding with macromolecules.

    PubMed

    Islam, M; Khanin, M; Sadik, O A

    2003-01-01

    Metals and radionuclides are usually coupled with proteins together with suitable ligands for therapeutic, tumor-imaging, pharmaceuticals, and biocompatibility applications. Several ligands that can strongly coordinate a given nuclide in a specific valency are already known. However, the demand for bifunctionality has limited the applications of these ligands. We hereby report the molecular design of a receptor system based on the linkage of protein to monoazo ligands. By use of basic coordination chemistry, 4-(3-quinolinoazo)hydroxybenzoic acid (QABA) and derivatives were successfully conjugated to ovalbumin, bovine serum albumin, and alkaline phosphatase at a site that was distinct from the metal binding site. The presence of carboxylic acid linkage in the QABA served as a convenient bridge for protein conjugation and may allow the generic application of these ligands for bioconjugate synthesis while ensuring a high in vivo stability. The ligand-protein conjugates were characterized using UV-vis spectroscopy, Fourier transform infrared spectroscopy, thin layer chromatography, NMR, and surface-enhanced laser desorption ionization time-of-flight mass spectrometry. The conjugate was tested for the ability to recognize nonradioactive Ga(3+) at a physiological pH, and a binding constant of 1 x 10(20) was recorded. Also, the in vitro testing results indicated that the fluorescent conjugates exhibited significant selectivity for gallium compared to Pb(2+), Hg(2+), Zn(2+), Cu(2+), Fe(3+), and Co(2+) while no responses were obtained for alkaline and alkaline earth metals. These attributes could allow these conjugates to be used as a model for imaging sensors and for metal detection. PMID:12523855

  8. Three bisphosphonate ligands improve the water solubility of quantum dots.

    PubMed

    Abdul Ghani, Siti Fatimah; Wright, Michael; Paramo, Juan Gallo; Bottrill, Melanie; Green, Mark; Long, Nicholas; Thanou, Maya

    2014-01-01

    Synthesised Quantum Dots (QDs) require surface modification in order to improve their aqueous dispersion and biocompatibility. Here, we suggest bisphosphonate molecules as agents to modify the surface of QDs for improved water solubility and biocompatibility. QDs_TOPO (CdSe/ZnS-trioctylphosphine oxide) were synthesised following modification of the method of Bawendi et al. (J. Phys. Chem. B, 1997, 101, 9463-9475). QDs surface modification is performed using a ligand exchange reaction with structurally different bisphosphonates (BIPs). The BIPs used were ethylene diphosphonate (EDP), methylenediphosphonate (MDP) and imidodiphosphonate (IDP). After ligand exchange, the QDs were extensively purified using centrifugation, PD-10 desalting columns and mini dialysis filters. Transmission electron microscopy (TEM) and fluorescent spectroscopy have been used to characterise the size and optical properties of the QDs. Cell toxicity was investigated using MTT (tetrazolium salt) and glutathione assays and intracellular uptake was imaged using confocal laser scanning microscopy and assessed by Inductively Coupled Plasma Mass Spectrometry (ICP-MS). QDs_TOPO and QDs-capped with BIPs (QDs_BIPs) were successfully synthesised. TEM showed the size and morphology of the QDs to be 5-7 nm with spherical shape. The stabilised QDs_BIPs showed significantly improved dispersion in aqueous solutions compared to QDs_TOPO. The cytotoxicity studies showed very rapid cell death for cells treated by QDs_TOPO and a minor effect on cell viability when QDs_BIPs were applied to the cells. Both EDP- and MDP-modified QDs did not significantly increase the intracellular levels of glutathione. In contrast, IDP-modified QDs substantially increased the intracellular glutathione levels, indicating potential cadmium leakage and inability of IDP to adequately cap and stabilise the QDs. EDP- and MDP-modified QDs were taken up by IGROV-1 (ovarian cancer) cells as shown by fluorescence microscopy, however, the

  9. Protein-ligand-based pharmacophores: generation and utility assessment in computational ligand profiling.

    PubMed

    Meslamani, Jamel; Li, Jiabo; Sutter, Jon; Stevens, Adrian; Bertrand, Hugues-Olivier; Rognan, Didier

    2012-04-23

    Ligand profiling is an emerging computational method for predicting the most likely targets of a bioactive compound and therefore anticipating adverse reactions, side effects and drug repurposing. A few encouraging successes have already been reported using ligand 2-D similarity searches and protein-ligand docking. The current study describes the use of receptor-ligand-derived pharmacophore searches as a tool to link ligands to putative targets. A database of 68,056 pharmacophores was first derived from 8,166 high-resolution protein-ligand complexes. In order to limit the number of queries, a maximum of 10 pharmacophores was generated for each complex according to their predicted selectivity. Pharmacophore search was compared to ligand-centric (2-D and 3-D similarity searches) and docking methods in profiling a set of 157 diverse ligands against a panel of 2,556 unique targets of known X-ray structure. As expected, ligand-based methods outperformed, in most of the cases, structure-based approaches in ranking the true targets among the top 1% scoring entries. However, we could identify ligands for which only a single method was successful. Receptor-ligand-based pharmacophore search is notably a fast and reliable alternative to docking when few ligand information is available for some targets. Overall, the present study suggests that a workflow using the best profiling method according to the protein-ligand context is the best strategy to follow. We notably present concrete guidelines for selecting the optimal computational method according to simple ligand and binding site properties. PMID:22480372

  10. Inducible fluorescent speckle microscopy

    PubMed Central

    Aguiar, Paulo; Belsley, Michael; Maiato, Helder

    2016-01-01

    The understanding of cytoskeleton dynamics has benefited from the capacity to generate fluorescent fiducial marks on cytoskeleton components. Here we show that light-induced imprinting of three-dimensional (3D) fluorescent speckles significantly improves speckle signal and contrast relative to classic (random) fluorescent speckle microscopy. We predict theoretically that speckle imprinting using photobleaching is optimal when the laser energy and fluorophore responsivity are related by the golden ratio. This relation, which we confirm experimentally, translates into a 40% remaining signal after speckle imprinting and provides a rule of thumb in selecting the laser power required to optimally prepare the sample for imaging. This inducible speckle imaging (ISI) technique allows 3D speckle microscopy to be performed in readily available libraries of cell lines or primary tissues expressing fluorescent proteins and does not preclude conventional imaging before speckle imaging. As a proof of concept, we use ISI to measure metaphase spindle microtubule poleward flux in primary cells and explore a scaling relation connecting microtubule flux to metaphase duration. PMID:26783303

  11. Fluorescence and Light Scattering

    ERIC Educational Resources Information Center

    Clarke, Ronald J.; Oprysa, Anna

    2004-01-01

    The aim of the mentioned experiment is to aid students in developing tactics for distinguishing between signals originating from fluorescence and light scattering. Also, the experiment provides students with a deeper understanding of the physicochemical bases of each phenomenon and shows that the techniques are actually related.

  12. Ultraviolet fluorescence monitor

    SciTech Connect

    Hargis, P.J. Jr.; Preppernau, B.L.; Aragon, B.P.

    1997-05-01

    A multispectral ultraviolet (UV) fluorescence imaging fluorometer and a pulsed molecular beam laser fluorometer were developed to detect volatile organic compounds of interest in environmental monitoring and drug interdiction applications. The UV fluorescence imaging fluorometer is a relatively simple instrument which uses multiple excitation wavelengths to measure the excitation/emission matrix for irradiated samples. Detection limits in the high part-per-million to low part-per-million range were measured for a number of volatile organic vapors in the atmosphere. Detection limits in the low part-per-million range were obtained using cryogenic cooling to pre-concentrate unknown samples before introducing them into the imaging fluorometer. A multivariate analysis algorithm was developed to analyze the excitation/emission matrix and used to determine the relative concentrations of species in computer synthesized mixtures containing up to five organic compounds. Analysis results demonstrated the utility of multispectral UV fluorescence in analytical measurements. A transportable UV fluorescence imaging fluorometer was used in two field tests. Field test results demonstrated that detection limits in the part-per-billion range were needed to reliably identify volatile organic compounds in realistic field test measurements. The molecular beam laser fluorometer, a more complex instrument with detection limits in the part-per-billion to part-per-trillion range, was therefore developed to satisfy detection sensitivity requirements for field test measurements. High-resolution spectroscopic measurements made with the molecular beam laser fluorometer demonstrated its utility in identifying volatile organic compounds in the atmosphere.

  13. Inducible fluorescent speckle microscopy.

    PubMed

    Pereira, António J; Aguiar, Paulo; Belsley, Michael; Maiato, Helder

    2016-01-18

    The understanding of cytoskeleton dynamics has benefited from the capacity to generate fluorescent fiducial marks on cytoskeleton components. Here we show that light-induced imprinting of three-dimensional (3D) fluorescent speckles significantly improves speckle signal and contrast relative to classic (random) fluorescent speckle microscopy. We predict theoretically that speckle imprinting using photobleaching is optimal when the laser energy and fluorophore responsivity are related by the golden ratio. This relation, which we confirm experimentally, translates into a 40% remaining signal after speckle imprinting and provides a rule of thumb in selecting the laser power required to optimally prepare the sample for imaging. This inducible speckle imaging (ISI) technique allows 3D speckle microscopy to be performed in readily available libraries of cell lines or primary tissues expressing fluorescent proteins and does not preclude conventional imaging before speckle imaging. As a proof of concept, we use ISI to measure metaphase spindle microtubule poleward flux in primary cells and explore a scaling relation connecting microtubule flux to metaphase duration. PMID:26783303

  14. Fluorescent Gage Indication

    NASA Technical Reports Server (NTRS)

    Barns, C. E.; Gilbaugh, B. L.; Gin, B.; Holt, W. L.; Lesak, P.; Mancini, R.; Spencer, H. F.

    1985-01-01

    Transfer of dye shows quality of contact between two mating parts. Mating parts checked for fit by spreading fluorescent dye on one, making brief light contact with other, and looking (under UV light) for transferred dye. Dye offers greater visibility under ultraviolet illumination, allowing better indication of how precisely parts match and what areas interfere.

  15. Fluorescence Imaging in Surgery

    PubMed Central

    Orosco, Ryan K.; Tsien, Roger Y.; Nguyen, Quyen T.

    2013-01-01

    Although the modern surgical era is highlighted by multiple technological advances and innovations, one area that has remained constant is the dependence of the surgeon's vision on white-light reflectance. This renders different body tissues in a limited palette of various shades of pink and red, thereby limiting the visual contrast available to the operating surgeon. Healthy tissue, anatomic variations, and diseased states are seen as slight discolorations relative to each other and differences are inherently limited in dynamic range. In the upcoming years, surgery will undergo a paradigm shift with the use of targeted fluorescence imaging probes aimed at augmenting the surgical armamentarium by expanding the “visible” spectrum available to surgeons. Such fluorescent “smart probes” will provide real-time, intraoperative, pseudo-color, high-contrast delineation of both normal and pathologic tissues. Fluorescent surgical molecular guidance promises another major leap forward to improve patient safety and clinical outcomes, and to reduce overall healthcare costs. This review provides an overview of current and future surgical applications of fluorescence imaging in diseased and nondiseased tissues and focus on the innovative fields of image processing and instrumentation. PMID:23335674

  16. Bimolecular fluorescence complementation.

    PubMed

    Wong, Katy A; O'Bryan, John P

    2011-01-01

    Defining the subcellular distribution of signaling complexes is imperative to understanding the output from that complex. Conventional methods such as immunoprecipitation do not provide information on the spatial localization of complexes. In contrast, BiFC monitors the interaction and subcellular compartmentalization of protein complexes. In this method, a fluororescent protein is split into amino- and carboxy-terminal non-fluorescent fragments which are then fused to two proteins of interest. Interaction of the proteins results in reconstitution of the fluorophore (Figure 1). A limitation of BiFC is that once the fragmented fluorophore is reconstituted the complex is irreversible. This limitation is advantageous in detecting transient or weak interactions, but precludes a kinetic analysis of complex dynamics. An additional caveat is that the reconstituted flourophore requires 30min to mature and fluoresce, again precluding the observation of real time interactions. BiFC is a specific example of the protein fragment complementation assay (PCA) which employs reporter proteins such as green fluorescent protein variants (BiFC), dihydrofolate reductase, b-lactamase, and luciferase to measure protein:protein interactions. Alternative methods to study protein:protein interactions in cells include fluorescence co-localization and Förster resonance energy transfer (FRET). For co-localization, two proteins are individually tagged either directly with a fluorophore or by indirect immunofluorescence. However, this approach leads to high background of non-interacting proteins making it difficult to interpret co-localization data. In addition, due to the limits of resolution of confocal microscopy, two proteins may appear co-localized without necessarily interacting. With BiFC, fluorescence is only observed when the two proteins of interest interact. FRET is another excellent method for studying protein:protein interactions, but can be technically challenging. FRET

  17. PDZ Affinity Chromatography: A general method for affinity purification of proteins based on PDZ domains and their ligands

    PubMed Central

    Walkup, Ward G.; Kennedy, Mary B.

    2014-01-01

    PDZ (PSD-95, DiscsLarge, ZO1) domains function in nature as protein binding domains within scaffold and membrane-associated proteins. They comprise ~ 90 residues and make specific, high affinity interactions with complementary C-terminal peptide sequences, with other PDZ domains, and with phospholipids. We hypothesized that the specific, strong interactions of PDZ domains with their ligands would make them well suited for use in affinity chromatography. Here we describe a novel affinity chromatography method applicable for the purification of proteins that contain PDZ domain-binding ligands, either naturally or introduced by genetic engineering. We created a series of affinity resins comprised of PDZ domains from the scaffold protein PSD-95, or from neuronal nitric oxide synthase (nNOS), coupled to solid supports. We used them to purify heterologously expressed neuronal proteins or protein domains containing endogenous PDZ domain ligands, eluting the proteins with free PDZ domain peptide ligands. We show that Proteins of Interest (POIs) lacking endogenous PDZ domain ligands can be engineered as fusion products containing C-terminal PDZ domain ligand peptides or internal, N- or C-terminal PDZ domains and then can be purified by the same method. Using this method, we recovered recombinant GFP fused to a PDZ-domain ligand in active form as verified by fluorescence yield. Similarly, chloramphenicol acetyltransferase (CAT) and β-Galactosidase (LacZ) fused to a C-terminal PDZ domain ligand or an N-terminal PDZ domain were purified in active form as assessed by enzymatic assay. In general, PDZ domains and ligands derived from PSD-95 were superior to those from nNOS for this method. PDZ Domain Affinity Chromatography promises to be a versatile and effective method for purification of a wide variety of natural and recombinant proteins. PMID:24607360

  18. Ligand-protected gold clusters: the structure, synthesis and applications

    NASA Astrophysics Data System (ADS)

    Pichugina, D. A.; Kuz'menko, N. E.; Shestakov, A. F.

    2015-11-01

    Modern concepts of the structure and properties of atomic gold clusters protected by thiolate, selenolate, phosphine and phenylacetylene ligands are analyzed. Within the framework of the superatom theory, the 'divide and protect' approach and the structure rule, the stability and composition of a cluster are determined by the structure of the cluster core, the type of ligands and the total number of valence electrons. Methods of selective synthesis of gold clusters in solution and on the surface of inorganic composites based, in particular, on the reaction of Aun with RS, RSe, PhC≡C, Hal ligands or functional groups of proteins, on stabilization of clusters in cavities of the α-, β and γ-cyclodextrin molecules (Au15 and Au25) and on anchorage to a support surface (Au25/SiO2, Au20/C, Au10/FeOx) are reviewed. Problems in this field are also discussed. Among the methods for cluster structure prediction, particular attention is given to the theoretical approaches based on the density functional theory (DFT). The structures of a number of synthesized clusters are described using the results obtained by X-ray diffraction analysis and DFT calculations. A possible mechanism of formation of the SR(AuSR)n 'staple' units in the cluster shell is proposed. The structure and properties of bimetallic clusters MxAunLm (M=Pd, Pt, Ag, Cu) are discussed. The Pd or Pt atom is located at the centre of the cluster, whereas Ag and Cu atoms form bimetallic compounds in which the heteroatom is located on the surface of the cluster core or in the 'staple' units. The optical properties, fluorescence and luminescence of ligand-protected gold clusters originate from the quantum effects of the Au atoms in the cluster core and in the oligomeric SR(AuSR)x units in the cluster shell. Homogeneous and heterogeneous reactions catalyzed by atomic gold clusters are discussed in the context of the reaction mechanism and the nature of the active sites. The bibliography includes 345 references.

  19. Fluorescence Microscopy of Single Molecules

    ERIC Educational Resources Information Center

    Zimmermann, Jan; van Dorp, Arthur; Renn, Alois

    2004-01-01

    The investigation of photochemistry and photophysics of individual quantum systems is described with the help of a wide-field fluorescence microscopy approach. The fluorescence single molecules are observed in real time.

  20. Improving protein-ligand docking with flexible interfacial water molecules using SWRosettaLigand.

    PubMed

    Li, Linqing; Xu, Weiwei; Lü, Qiang

    2015-11-01

    Computational protein-ligand docking is of great importance in drug discovery and design. Conformational changes greatly affect the results of protein-ligand docking, especially when water molecules take part in mediating protein ligand interactions or when large conformational changes are observed in the receptor backbone interface. We have developed an improved protocol, SWRosettaLigand, based on the RosettaLigand protocol. This approach incorporates the flexibility of interfacial water molecules and modeling of the interface of the receptor into the original RosettaLigand. In a coarse sampling step, SWRosettaLigand pre-optimizes the initial position of the water molecules, docks the ligand to the receptor with explicit water molecules, and minimizes the predicted structure with water molecules. The receptor backbone interface is treated as a loop and perturbed and refined by kinematic closure, or cyclic coordinate descent algorithm, with the presence of the ligand. In two cross-docking test sets, it was identified that for 8 out of 14, and 16 out of 22, test instances, the top-ranked structures by SWRosettaLigand achieved better accuracy than other protocols. PMID:26515196

  1. Strep-tag II Mutant Maltose-binding Protein for Reagentless Fluorescence Sensing

    PubMed Central

    Hasmoni, Siti Halimah; Mau, Goh Kian; Karsani, Saiful Anuar; Cass, Anthony; Shahir, Shafinaz

    2016-01-01

    Maltose-binding protein (MBP) is a periplasmic binding protein found in Gram negative bacteria. MBP is involved in maltose transport and bacterial chemotaxis; it binds to maltose and maltodextrins comprising α(1–4)-glucosidically linked linear glucose polymers and α(1–4)-glucosidically linked cyclodextrins. Upon ligand binding, MBP changes its conformation from an open to a closed form. This molecular recognition—transducing a ligand-binding event into a physical one—renders MBP an ideal candidate for biosensor development. Here, we describe the construction of a Strep-tag II mutant MBP for reagentless fluorescence sensing. malE, which encodes MBP, was amplified. A cysteine residue was introduced by site-directed mutagenesis to ensure a single label attachment at a specific site with a thiol-specific fluorescent probe. An environmentally sensitive fluorophore (IANBD amide) was covalently attached to the introduced thiol group and analysed by fluorescence sensing. The tagged mutant MBP (D95C) was purified (molecular size, ∼42 kDa). The fluorescence measurements of the IANBD-labelled Strep-tag II–D95C in the solution phase showed an appreciable change in fluorescence intensity (dissociation constant, 7.6±1.75 μM). Our mutant MBP retains maltose-binding activity and is suitable for reagentless fluorescence sensing. PMID:27019682

  2. A mechanosynthesized, sequential, cyclic fluorescent probe for mercury and iodide ions in aqueous solutions

    NASA Astrophysics Data System (ADS)

    Chen, Shangwen; Wang, Pipi; Jia, Chunmei; Lin, Qiang; Yuan, Wenbing

    2014-12-01

    A fluorescent Hg2+-selective chemosensor, 2,5-dimethoxybenzaldehyde thiosemicarbazone (1), was quantitatively prepared by grinding 2,5-dimethoxybenzaldehyde and thiosemicarbazide together in a ball mill for 15 min. The excitation and emission maxima of compound 1 are 347 and 450 nm, respectively. The reaction of this ligand with Hg2+ was investigated by FT-IR, 1H NMR, and fluorescence titration. Results show that the composition of the resulting Hg complex 1-Hg is 2:1 1:Hg, and that the S and imino N atoms serve as the binding sites of the ligand to the Hg2+ ions. Coordination-assisted fluorescence quenching results show that compound 1 exhibits a highly selective fluorescence response to trace amounts of Hg2+ in water. More importantly, the resulting complex 1-Hg can be used as a turn-on fluorescence probe for I- at a detection limit of 8.4 × 10-8 M. Thus, compound 1 is a relatively stable, sequential, cyclic fluorescent probe for Hg2+ and I-.

  3. A mechanosynthesized, sequential, cyclic fluorescent probe for mercury and iodide ions in aqueous solutions.

    PubMed

    Chen, Shangwen; Wang, Pipi; Jia, Chunmei; Lin, Qiang; Yuan, Wenbing

    2014-12-10

    A fluorescent Hg(2+)-selective chemosensor, 2,5-dimethoxybenzaldehyde thiosemicarbazone (1), was quantitatively prepared by grinding 2,5-dimethoxybenzaldehyde and thiosemicarbazide together in a ball mill for 15min. The excitation and emission maxima of compound 1 are 347 and 450nm, respectively. The reaction of this ligand with Hg(2+) was investigated by FT-IR, (1)H NMR, and fluorescence titration. Results show that the composition of the resulting Hg complex 1-Hg is 2:1 1:Hg, and that the S and imino N atoms serve as the binding sites of the ligand to the Hg(2+) ions. Coordination-assisted fluorescence quenching results show that compound 1 exhibits a highly selective fluorescence response to trace amounts of Hg(2+) in water. More importantly, the resulting complex 1-Hg can be used as a turn-on fluorescence probe for I(-) at a detection limit of 8.4×10(-8)M. Thus, compound 1 is a relatively stable, sequential, cyclic fluorescent probe for Hg(2+) and I(-). PMID:24945863

  4. Direct evidence of energy transfer from a singlet ligand level to lanthanide ions in their diketonate complexes

    NASA Astrophysics Data System (ADS)

    Mironov, L. Yu.; Sveshnikova, E. B.; Ermolaev, V. L.

    2015-07-01

    We have compared the fluorescence intensity of 2-naphthoyltrifluoroacetonate (NTA) in nanoparticles from Gd(NTA)3phen complexes with the fluorescence intensities of this compound in nanoparticles from similar complexes of Pr, Nd, Sm, Eu, Tb, Dy, Er, Ho, and Tm, which absorb in the NTA fluorescence range. We have proven that there is energy transfer from the S 1 level of NTA ligands to Ln(III) ions, which occurs with rates k tr ˜ 1011-1012 s-1. We have also studied the competition between two processes: energy transfer from Ln(NTA)3phen to Ln(III) ions and energy transfer to Nile blue molecules incorporated into nanoparticles from complexes of these ions. It has been shown that, in nanoparticles from complexes of Nd(III), Tb(III), Dy(III), and Tm(III) ions, which are incapable of sensitizing the fluorescence of Nile blue, the values of k tr2 for the energy transfer from NTA to Ln(III) ions, which were obtained from the data on the change in the intensity of the sensitized fluorescence of Nile blue, completely coincide with the values of k tr1 determined from the fluorescence quenching of NTA in these nanoparticles. We have found that, in nanoparticles from Pr, Sm, Eu, Er, and Ho complexes, the efficiency of the sensitized fluorescence of Nile blue is higher than that predicted from the fluorescence quenching of NTA by these ions in their complexes, which indicates that all these ions participate as mediators in the energy transfer from ligands to the dye.

  5. Ruthenium(II) polypyridyl complexes with hydrophobic ancillary ligand as Aβ aggregation inhibitors.

    PubMed

    Vyas, Nilima A; Ramteke, Shefali N; Kumbhar, Avinash S; Kulkarni, Prasad P; Jani, Vinod; Sonawane, Uddhavesh B; Joshi, Rajendra R; Joshi, Bimba; Erxleben, Andrea

    2016-10-01

    The synthesis, spectral and electrochemical characterization of the complexes of the type [Ru(NN)2(txbg)](2+) where NN is 2,2'-bipyridine (bpy) (1), 1,10-phenanthroline (phen) (2), dipyrido [3,2-d:2',3f] quinoxaline (dpq) (3), and dipyrido[3,2-a:2',3'-c]phenazine (dppz) (4) which incorporate the tetra-xylene bipyridine glycoluril (txbg) as the ancillary ligand are described in detail. Crystal structures of ligand txbg and complex 2 were solved by single crystal X-ray diffraction. Thioflavin T (ThT) fluorescence and Transmission Electron Microscopy (TEM) results indicated that at micromolar concentration all complexes exhibit significant potential of Aβ aggregation inhibition, while the ligand txbg displayed weak activity towards Aβ aggregation. Complex 1 showed relatively low inhibition (70%) while complexes 2-4 inhibited nearly 100% Aβ aggregation after 240 h of incubation. The similar potential of complexes 2-4 and absence of any trend in their activity with the planarity of polypyridyl ligands suggests there is no marked effect of planarity of coligands on their inhibitory potential. Further studies on acetylcholinesterase (AChE) inhibition indicated very weak activity of these complexes against AChE. Detailed interactions of Aβ with both ligand and complex 2 have been studied by molecular modeling. Complex 2 showed interactions involving all three polypyridyl ligands with hydrophobic region of Aβ. Furthermore, the toxicity of these complexes towards human neuroblastoma cells was evaluated by MTT assay and except complex 4, the complexes displayed very low toxicity. PMID:27406812

  6. Ultrafast dynamics of ligands within heme proteins.

    PubMed

    Vos, Marten H

    2008-01-01

    Physiological bond formation and bond breaking events between proteins and ligands and their immediate consequences are difficult to synchronize and study in general. However, diatomic ligands can be photodissociated from heme, and thus in heme proteins ligand release and rebinding dynamics and trajectories have been studied on timescales of the internal vibrations of the protein that drive many biochemical reactions, and longer. The rapidly expanding number of characterized heme proteins involved in a large variety of functions allows comparative dynamics-structure-function studies. In this review, an overview is given of recent progress in this field, and in particular on initial sensing processes in signaling proteins, and on ligand and electron transfer dynamics in oxidases and cytochromes. PMID:17996720

  7. The Retinoid X Receptors and Their Ligands

    PubMed Central

    Dawson, Marcia I.; Xia, Zebin

    2014-01-01

    This chapter presents an overview of the current status of studies on the structural and molecular biology of the retinoid X receptor subtypes α, β, and γ (RXRs, NR2B1–3), their nuclear and cytoplasmic functions, post-transcriptional processing, and recently reported ligands. Points of interest are the different changes in the ligand-binding pocket induced by variously shaped agonists, the communication of the ligand–bound pocket with the coactivator binding surface and the heterodimerization interface, and recently identified ligands that are natural products, those that function as environmental toxins or drugs that had been originally designed to interact with other targets, as well as those that were deliberately designed as RXR-selective transcriptional agonists, synergists, or antagonists. Of these synthetic ligands, the general trend in design appears to be away from fully aromatic rigid structures to those containing partial elements of the flexible tetraene side chain of 9-cis-retinoic acid. PMID:22020178

  8. Bioisosteric matrices for ligands of serotonin receptors.

    PubMed

    Warszycki, Dawid; Mordalski, Stefan; Staroń, Jakub; Bojarski, Andrzej J

    2015-04-01

    The concept of bioisosteric replacement matrices is applied to explore the chemical space of serotonin receptor ligands, aiming to determine the most efficient ways of manipulating the affinity for all 5-HT receptor subtypes. Analysis of a collection of over 1 million bioisosteres of compounds with measured activity towards serotonin receptors revealed that an average of 31 % of the ligands for each target are mutual bioisosteres. In addition, the collected dataset allowed the development of bioisosteric matrices-qualitative and quantitative descriptions of the biological effects of each predefined type of bioisosteric substitution, providing favored paths of modifying the compounds. The concept exemplified here for serotonin receptor ligands can likely be more broadly applied to other target classes, thus representing a useful guide for medicinal chemists designing novel ligands. PMID:25772514

  9. Ligand engineering of nanoparticle solar cells

    NASA Astrophysics Data System (ADS)

    Voros, Marton

    Semiconductor nanoparticles (NP) are promising materials to build cheap and efficient solar cells. One of the key challenges in their utilization for solar energy conversion is the control of NP surfaces and ligand-NP interfaces. Recent experiments have shown that by carefully choosing the ligands terminating the NPs, one can tailor electronic and optical absorption properties of NP assemblies, along with their transport properties. By using density functional theory based methods, we investigated how the opto-electronic properties of lead chalcogenide NPs may be tuned by using diverse organic and inorganic ligands. We interpreted experiments, and we showed that an essential prerequisite to avoid detrimental trap states is to ensure charge balance at the ligand-NP interface, possibly with the help of hydrogen treatment Work supported by the Center for Advanced Solar Photophysics, an Energy Frontier Research Center funded by the US Department of Energy, Office of Science, Office of Basic Energy Sciences.

  10. Substitution Pattern Reverses the Fluorescence Response of Coumarin Glycoligands upon Coordination with Silver (I)

    PubMed Central

    Shi, De-Tai; Wei, Xiao-Li; Sheng, Yayun; Zang, Yi; He, Xiao-Peng; Xie, Juan; Liu, Guixia; Tang, Yun; Li, Jia; Chen, Guo-Rong

    2014-01-01

    Development of sugar-based fluorescence (FL) chemo-probes is of much interest since sugars are biocompatible, water-soluble and structurally rigid natural starting materials. We report here that fluorescent glycoligands with two triazolyl coumarin moieties installed onto the different positions of an identical glucosyl nucleus exert completely reversed optical response to a metal ion. C3,4-, C2,3- and C4,6-di-substituted coumarin glucosides synthesized by a click reaction similarly showed a selective FL variation in the presence of silver (I) among a range of metal cations in an aqueous solution. However, the variation was determined to be converse: the FL of the C3,4-ligand was quenched whereas that of the C2,3/C4,6-ligand tangibly enhanced. FL and NMR titrations suggested that this divergence was due to the distinct complexation modes of the conformationally constrained ligands with the ion. The optimal motifs of the ligand-ion complexation were predicted by a computational simulation. Finally, the C2,3-ligand was determined to be of low cytotoxicity and applicable in the FL imaging of silver ions internalized by live cells. PMID:24584644

  11. Substitution Pattern Reverses the Fluorescence Response of Coumarin Glycoligands upon Coordination with Silver (I)

    NASA Astrophysics Data System (ADS)

    Shi, De-Tai; Wei, Xiao-Li; Sheng, Yayun; Zang, Yi; He, Xiao-Peng; Xie, Juan; Liu, Guixia; Tang, Yun; Li, Jia; Chen, Guo-Rong

    2014-03-01

    Development of sugar-based fluorescence (FL) chemo-probes is of much interest since sugars are biocompatible, water-soluble and structurally rigid natural starting materials. We report here that fluorescent glycoligands with two triazolyl coumarin moieties installed onto the different positions of an identical glucosyl nucleus exert completely reversed optical response to a metal ion. C3,4-, C2,3- and C4,6-di-substituted coumarin glucosides synthesized by a click reaction similarly showed a selective FL variation in the presence of silver (I) among a range of metal cations in an aqueous solution. However, the variation was determined to be converse: the FL of the C3,4-ligand was quenched whereas that of the C2,3/C4,6-ligand tangibly enhanced. FL and NMR titrations suggested that this divergence was due to the distinct complexation modes of the conformationally constrained ligands with the ion. The optimal motifs of the ligand-ion complexation were predicted by a computational simulation. Finally, the C2,3-ligand was determined to be of low cytotoxicity and applicable in the FL imaging of silver ions internalized by live cells.

  12. Holograms of fluorescent albumin

    NASA Astrophysics Data System (ADS)

    Ordóñez-Padilla, M. J.; Olivares-Pérez, A.; Berriel-Valdos, L. R.; Mejias-Brizuela, N. Y.; Fuentes-Tapia, I.

    2011-09-01

    We report the characterization and analysis of photochromic films gallus gallus albumin as a matrix modified for holographic recording. Photo-oxidation of homogeneous mixtures prepared with albumin-propylene glycol, to combine chemically with aqueous solution of ammonium dichromate at certain concentrations. We analyzed the diffraction gratings, through the diffraction efficiency of the proposed material. Also, eosin was used as a fluorescent agent, so it is found that produces an inhibitory effect, thus decreasing the diffraction efficiency of the matrices prepared in near-identical circumstances. The work was to achieve stability of albumin films, were prepared with propylene glycol. Finally, experimental studies were performed with films when subjected to aqueous solution of eosin (fluorescent agent) to verify the ability to increase or decrease in diffraction efficiency.

  13. Integrated fluorescence analysis system

    DOEpatents

    Buican, Tudor N.; Yoshida, Thomas M.

    1992-01-01

    An integrated fluorescence analysis system enables a component part of a sample to be virtually sorted within a sample volume after a spectrum of the component part has been identified from a fluorescence spectrum of the entire sample in a flow cytometer. Birefringent optics enables the entire spectrum to be resolved into a set of numbers representing the intensity of spectral components of the spectrum. One or more spectral components are selected to program a scanning laser microscope, preferably a confocal microscope, whereby the spectrum from individual pixels or voxels in the sample can be compared. Individual pixels or voxels containing the selected spectral components are identified and an image may be formed to show the morphology of the sample with respect to only those components having the selected spectral components. There is no need for any physical sorting of the sample components to obtain the morphological information.

  14. Glutamate receptor ligands as anxiolytics.

    PubMed

    Chojnacka-Wójcik, E; Kłodzinska, A; Pilc, A

    2001-08-01

    The glutamatergic system has received considerable attention over recent years as a potential target for anxiolytic drugs. In spite of the pronounced anxiolytic-like effects of competitive and non-competitive antagonists of NMDA receptors in animal models of anxiety, these substances can not be regarded as potential anxiolytic drugs, mainly due to their side-effect profiles (eg, ataxia, myorelaxation, impairment of learning and memory processes and psychotomimetic effects). Antagonists and partial agonists of the glycine, receptor inhibit function of the NMDA receptor complex and evoke in animals an anxiolytic-like response. Although data concerning anti-anxiety-like effects of glycine, receptor antagonists are not very promising, studies are underway to develop new, brain-penetrating agents devoid of side effects. Further developments are necessary to more fully elucidate the possible involvement of AMPA/kainate receptors in anxiety. The recent discovery of metabotropic glutamate receptors, which modulate the function of the glutamatergic system, offers new hope for discovery of a new generation of anxiolytics. MPEP, a highly selective, brain penetrable, noncompetitive mGlu5 receptor antagonist, evokes anxiolytic-like effects in several animal models of anxiety, remaining remarkably free of side effects. LY-354740, a selective brain-penetrable group II mGlu receptor agonist, evokes marked anxiolytic-like effects in animal models of anxiety. LY-354740 causes mild sedation in mice, does not disturb motor coordination and has no potential to cause dependence. Therefore mGlu receptor ligands may become the anxiolytics of the future, free from the side effects characteristic of benzodiazepines. PMID:11892923

  15. Synthesis and spectral studies of copper complexes using a N-octylated bis benzimidazole diamide ligand

    NASA Astrophysics Data System (ADS)

    Mohapatra, Subash Chandra; Mathur, Pavan

    2011-02-01

    Monomeric Cu(II) and Cu(I) complexes bound to a tetradentate bis-benzimidazole diamide ligand N,N'-bis(N-octyl benzimidazolyl-2yl)(methyl)pentane diamide (O-GBGA) have been isolated and characterized. X-Band EPR spectra of the copper(II) complexes in CH 2Cl 2 were recorded in a frozen solution as solvent at liquid nitrogen temperature. Solution spectra typically indicate a d ground state ( g|| > g⊥ > 2.0023) and show less than four nuclear hyperfine lines with broadening of g⊥ line in some cases, thus indicating distorted tetragonal geometry. One of the copper(II) complexes shows a five line N-SHF structure (16 ± 1 G) implying the binding of imine nitrogen of the benzimidazole to copper ion. α2 ranges from 0.57-0.97 indicating considerable amount of covalent character in Cu-L bond. Anodic shifts in E1/2 values indicate the retention of anion in the coordination sphere of Cu(II), E1/2 values becoming anodic in the order C 6H 5COO - < SCN - < Cl -. The fluorescence quantum yield of complexes was found to be lower than that of the ligand O-GBGA ( Φ = 0.029) and the relative fluorescence data reveals that fluorescence of such compounds could distinguish between small and large anions.

  16. Fluorescence biosensing in nanopores.

    PubMed

    Karolin, Jan; Pánek, Dalibor; MacMillan, Alexander; Rolinski, Olaf; Birch, David

    2009-01-01

    Hydrated nanopores offer a unique environment for studying biological molecules under controlled conditions and fabricating sensors using fluorescence. Silica nanopores for example are non-toxic, biologically and optically compatible with protein, and can be easily synthesized to entrap protein and exclude potentially interfering macromolecules, while transmitting analytes of interest. A well known problem when polymerizing orthosilicates to fabricate silica sol-gel nanopores is the release of alcohol, which denatures proteins. We will describe how using the fluorescence of PRODAN (6-propionyl-2-(N,N-dimethylamino) naphthalene) to monitor methanol generated during polymerization has helped define a protocol with enhanced biocompatibility. The improved biocompatibility of sol-gel nanopores synthesized using tetramethyl orthosilicate (TMOS) has been demonstrated by preserving the unstable native trimer form of allophycocyanin (APC) for up to 500 Hrs without the need to covalently binding the subunits together. This has enabled the observation of native APC trimer by means of its fluorescence in a pore down to the single molecule level. In this paper we demonstrate how PRODAN and another polarity sensitive dye, 9-diethylamino-5H-benzo[alpha]phenoxazine-5-one, Nile red (NR) report on pore polarity and successfully extend protein encapsulation to nano-channels of alumina (Al2O3). Improved biocompatibility of nanopores has potential impact in nanomedicine where the ability to study single biomolecules is a primary goal as it underpins our understanding of disease pathology and therapeutics at the most fundamental level. In sensing also the advantages of nanopore isolation of metabolite-specific protein for detecting non-fluorescent metabolites has been demonstrated. Similar approaches can in principle be developed for both single-molecules and lab-on-a-chip sensors. PMID:19964618

  17. Magnetic fluorescent lamp

    DOEpatents

    Berman, S.M.; Richardson R.W.

    1983-12-29

    The radiant emission of a mercury-argon discharge in a fluorescent lamp assembly is enhanced by providing means for establishing a magnetic field with lines of force along the path of electron flow through the bulb of the lamp assembly, to provide Zeeman splitting of the ultraviolet spectral line. Optimum results are obtained when the magnetic field strength causes a Zeeman splitting of approximately 1.7 times the thermal line width.

  18. Green fluorescent protein: A perspective

    PubMed Central

    Remington, S James

    2011-01-01

    A brief personal perspective is provided for green fluorescent protein (GFP), covering the period 1994–2011. The topics discussed are primarily those in which my research group has made a contribution and include structure and function of the GFP polypeptide, the mechanism of fluorescence emission, excited state protein transfer, the design of ratiometric fluorescent protein biosensors and an overview of the fluorescent proteins derived from coral reef animals. Structure-function relationships in photoswitchable fluorescent proteins and nonfluorescent chromoproteins are also briefly covered. PMID:21714025

  19. PLIC: protein-ligand interaction clusters.

    PubMed

    Anand, Praveen; Nagarajan, Deepesh; Mukherjee, Sumanta; Chandra, Nagasuma

    2014-01-01

    Most of the biological processes are governed through specific protein-ligand interactions. Discerning different components that contribute toward a favorable protein- ligand interaction could contribute significantly toward better understanding protein function, rationalizing drug design and obtaining design principles for protein engineering. The Protein Data Bank (PDB) currently hosts the structure of ∼68 000 protein-ligand complexes. Although several databases exist that classify proteins according to sequence and structure, a mere handful of them annotate and classify protein-ligand interactions and provide information on different attributes of molecular recognition. In this study, an exhaustive comparison of all the biologically relevant ligand-binding sites (84 846 sites) has been conducted using PocketMatch: a rapid, parallel, in-house algorithm. PocketMatch quantifies the similarity between binding sites based on structural descriptors and residue attributes. A similarity network was constructed using binding sites whose PocketMatch scores exceeded a high similarity threshold (0.80). The binding site similarity network was clustered into discrete sets of similar sites using the Markov clustering (MCL) algorithm. Furthermore, various computational tools have been used to study different attributes of interactions within the individual clusters. The attributes can be roughly divided into (i) binding site characteristics including pocket shape, nature of residues and interaction profiles with different kinds of atomic probes, (ii) atomic contacts consisting of various types of polar, hydrophobic and aromatic contacts along with binding site water molecules that could play crucial roles in protein-ligand interactions and (iii) binding energetics involved in interactions derived from scoring functions developed for docking. For each ligand-binding site in each protein in the PDB, site similarity information, clusters they belong to and description of

  20. Evaluation of Chemical Fluorescent Dyes as a Protein Conjugation Partner for Live Cell Imaging

    PubMed Central

    Hayashi-Takanaka, Yoko; Stasevich, Timothy J.; Kurumizaka, Hitoshi; Nozaki, Naohito; Kimura, Hiroshi

    2014-01-01

    To optimize live cell fluorescence imaging, the choice of fluorescent substrate is a critical factor. Although genetically encoded fluorescent proteins have been used widely, chemical fluorescent dyes are still useful when conjugated to proteins or ligands. However, little information is available for the suitability of different fluorescent dyes for live imaging. We here systematically analyzed the property of a number of commercial fluorescent dyes when conjugated with antigen-binding (Fab) fragments directed against specific histone modifications, in particular, phosphorylated H3S28 (H3S28ph) and acetylated H3K9 (H3K9ac). These Fab fragments were conjugated with a fluorescent dye and loaded into living HeLa cells. H3S28ph-specific Fab fragments were expected to be enriched in condensed chromosomes, as H3S28 is phosphorylated during mitosis. However, the degree of Fab fragment enrichment on mitotic chromosomes varied depending on the conjugated dye. In general, green fluorescent dyes showed higher enrichment, compared to red and far-red fluorescent dyes, even when dye∶protein conjugation ratios were similar. These differences are partly explained by an altered affinity of Fab fragment after dye-conjugation; some dyes have less effect on the affinity, while others can affect it more. Moreover, red and far-red fluorescent dyes tended to form aggregates in the cytoplasm. Similar results were observed when H3K9ac-specific Fab fragments were used, suggesting that the properties of each dye affect different Fab fragments similarly. According to our analysis, conjugation with green fluorescent dyes, like Alexa Fluor 488 and Dylight 488, has the least effect on Fab affinity and is the best for live cell imaging, although these dyes are less photostable than red fluorescent dyes. When multicolor imaging is required, we recommend the following dye combinations for optimal results: Alexa Fluor 488 (green), Cy3 (red), and Cy5 or CF640 (far-red). PMID:25184362

  1. Fluorescence in complexes based on quinolines-derivatives: a search for better fluorescent probes.

    PubMed

    Mecca, Carolina Z P; Fonseca, Fernando L A; Bagatin, Izilda A

    2016-11-01

    Quinoline-derived fluorescent complexes were designed; synthesized by the reaction of 5-nitro-8-hydroxyquinoline and 5-chloro-8-hydroxyquinoline with Al(3+), Mg(2+), Zn(2+), and Cd(2+) salts (1-8); and characterized. The (1)H NMR spectra of complexes 1 and 5, containing Al(3+), were consistent with an octahedral structure having approximate D3 symmetry, and the results supported the favored facial isomer (fac). Data for complexes 2-4 and 6-8 supported the formation of tetrahedral structures. Intense luminescence was detected for complexes 5-8, even with the naked eye, as indicated by quantum yield values of 0.087, 0.094, 0.051, and 0.021, respectively. Furthermore, in contrast to 5-nitro-8-hydroxyquinoline, the 5-chloro-8-hydroxyquinoline ligand exhibited bands at different energies depending on the coordinated metal, which supported its potential application in ionic and biological probes, as well as in cell imaging. PMID:27288961

  2. Synthesis and characterization of a fluorescent probe for α-tocopherol suitable for fluorescence microscopy.

    PubMed

    Ghelfi, Mikel; Ulatowski, Lynn; Manor, Danny; Atkinson, Jeffrey

    2016-06-15

    Previously prepared fluorescent derivatives of α-tocopherol have shown tremendous utility in both in vitro exploration of the mechanism of ligand transfer by the α-tocopherol transfer protein (α-TTP) and the intracellular transport of α-tocopherol in cells and tissues. We report here the synthesis of a 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) containing α-tocopherol analog having extended conjugation with an alkenyl thiophene group that extends the absorption and emission maxima to longer wavelengths (λex=571nm and λem=583nm). The final fluorophore thienyl-ene-BODIPY-α-tocopherol, 2, binds to recombinant human α-TTP with a Kd=8.7±1.1nM and is a suitable probe for monitoring the secretion of α-tocopherol from cultured Mcf7#189 cells. PMID:27161877

  3. Fluorescence in complexes based on quinolines-derivatives: a search for better fluorescent probes

    NASA Astrophysics Data System (ADS)

    Mecca, Carolina Z. P.; Fonseca, Fernando L. A.; Bagatin, Izilda A.

    2016-11-01

    Quinoline-derived fluorescent complexes were designed; synthesized by the reaction of 5-nitro-8-hydroxyquinoline and 5-chloro-8-hydroxyquinoline with Al3+, Mg2+, Zn2+, and Cd2+ salts (1-8); and characterized. The 1H NMR spectra of complexes 1 and 5, containing Al3+, were consistent with an octahedral structure having approximate D3 symmetry, and the results supported the favored facial isomer (fac). Data for complexes 2-4 and 6-8 supported the formation of tetrahedral structures. Intense luminescence was detected for complexes 5-8, even with the naked eye, as indicated by quantum yield values of 0.087, 0.094, 0.051, and 0.021, respectively. Furthermore, in contrast to 5-nitro-8-hydroxyquinoline, the 5-chloro-8-hydroxyquinoline ligand exhibited bands at different energies depending on the coordinated metal, which supported its potential application in ionic and biological probes, as well as in cell imaging.

  4. Cyanine-based probe\\tag-peptide pair fluorescence protein imaging and fluorescence protein imaging methods

    DOEpatents

    Mayer-Cumblidge, M. Uljana; Cao, Haishi

    2013-01-15

    A molecular probe comprises two arsenic atoms and at least one cyanine based moiety. A method of producing a molecular probe includes providing a molecule having a first formula, treating the molecule with HgOAc, and subsequently transmetallizing with AsCl.sub.3. The As is liganded to ethanedithiol to produce a probe having a second formula. A method of labeling a peptide includes providing a peptide comprising a tag sequence and contacting the peptide with a biarsenical molecular probe. A complex is formed comprising the tag sequence and the molecular probe. A method of studying a peptide includes providing a mixture containing a peptide comprising a peptide tag sequence, adding a biarsenical probe to the mixture, and monitoring the fluorescence of the mixture.

  5. A universal approach to ultrasmall magneto-fluorescent nanohybrids.

    PubMed

    Feld, Artur; Merkl, Jan-Philip; Kloust, Hauke; Flessau, Sandra; Schmidtke, Christian; Wolter, Christopher; Ostermann, Johannes; Kampferbeck, Michael; Eggers, Robin; Mews, Alf; Schotten, Theo; Weller, Horst

    2015-10-12

    Seeded emulsion polymerization is a powerful universal method to produce ultrasmall multifunctional magnetic nanohybrids. In a two-step procedure, iron oxide nanocrystals were initially encapsulated in a polystyrene (PS) shell and subsequently used as beads for a controlled assembly of elongated quantum dots/quantum rods (QDQRs). The synthesis of a continuous PS shell allows the whole construct to be fixed and the composition of the nanohybrid to be tuned. The fluorescence of the QDQRs and magnetism of iron oxide were perfectly preserved, as confirmed by single-particle investigation, fluorescence decay measurements, and relaxometry. Bio-functionalization of the hybrids was straightforward, involving copolymerization of appropriate affinity ligands as shown by immunoblot analysis. Additionally, the universality of this method was shown by the embedment of a broad scale of NPs. PMID:26136318

  6. A methacrylate-based polymeric imidazole ligand yields quantum dots with low cytotoxicity and low nonspecific binding

    PubMed Central

    Johnson, Colin M.; Pate, Kayla M.; Shen, Yi; Viswanath, Anand; Tan, Rui; Benicewicz, Brian C.; Moss, Melissa A.; Greytak, Andrew B.

    2016-01-01

    This paper assesses the biocompatibility for fluorescence imaging of colloidal nanocrystal quantum dots (QDs) coated with a recently-developed multiply-binding methacrylate-based polymeric imidazole ligand. The QD samples were purified prior to ligand exchange via a highly repeatable gel permeation chromatography (GPC) method. A multi-well plate based protocol was used to characterize nonspecific binding and toxicity of the QDs toward human endothelial cells. Nonspecific binding in 1% fetal bovine serum was negligible compared to anionically-stabilized QD controls, and no significant toxicity was detected on 24 h exposure. The nonspecific binding results were confirmed by fluorescence microscopy. This study is the first evaluation of biocompatibility in QDs initially purified by GPC and represents a scalable approach to comparison among nanocrystal-based bioimaging scaffolds. PMID:26247382

  7. Photodegradation of luminescence in organic-ligand-capped Eu{sup 3+}:LaF{sub 3} nano-particles

    SciTech Connect

    King, Gavin G. G.; Taylor, Luke R.; Longdell, Jevon J.; Clarke, David J.; Quilty, J. W.

    2014-01-28

    The luminescence from europium doped lanthanum trifluoride (Eu{sup 3+}:LaF{sub 3}) nano-crystals can be greatly enhanced by capping with β-diketonate organic ligands. Here, we report on photo-stability measurements for the case of nano-crystals capped with thenoyltrifluroacetone (TTA) and compared with those capped with an inactive ligand, oleic acid. With exposure to UV pump light, we observed significant decrease in fluorescence and change in emission spectrum of the TTA-capped nano-particles whilst the fluorescence lifetime remained approximately constant. After a dose of order 70 kJ cm{sup −2}, the luminescence level was similar to that of oleic acid capped nano-crystals. We discuss possible mechanisms.

  8. Fully Flexible Docking of Medium Sized Ligand Libraries with RosettaLigand

    PubMed Central

    DeLuca, Samuel; Khar, Karen; Meiler, Jens

    2015-01-01

    RosettaLigand has been successfully used to predict binding poses in protein-small molecule complexes. However, the RosettaLigand docking protocol is comparatively slow in identifying an initial starting pose for the small molecule (ligand) making it unfeasible for use in virtual High Throughput Screening (vHTS). To overcome this limitation, we developed a new sampling approach for placing the ligand in the protein binding site during the initial ‘low-resolution’ docking step. It combines the translational and rotational adjustments to the ligand pose in a single transformation step. The new algorithm is both more accurate and more time-efficient. The docking success rate is improved by 10–15% in a benchmark set of 43 protein/ligand complexes, reducing the number of models that typically need to be generated from 1000 to 150. The average time to generate a model is reduced from 50 seconds to 10 seconds. As a result we observe an effective 30-fold speed increase, making RosettaLigand appropriate for docking medium sized ligand libraries. We demonstrate that this improved initial placement of the ligand is critical for successful prediction of an accurate binding position in the ‘high-resolution’ full atom refinement step. PMID:26207742

  9. Fluorescence photodiagnosis in clinical practice.

    PubMed

    Moghissi, K; Stringer, M R; Dixon, Kate

    2008-12-01

    Fluorescence diagnosis has become an important method of investigation in clinical practice particularly in identification and localisation of pre and early cancerous lesions as well as image guided therapy. The method relies on the principle of differential fluorescence emission between abnormal and normal tissues in response to excitation by a specific wavelength of light within the visible spectrum range. In clinical practice two types of fluorescence diagnostic methods are used, namely autofluorescence and drug-induced fluorescence. The former relies on the differential fluorescence of "native" fluorophores whereas the latter requires a photosensitiser which enhances the differential fluorescence emission of the normal versus the abnormal tissues. Development and advances in fibreoptic, endoscopic instrumentation currently permit fluorescence endoscopy to be carried out in a number of situations. PMID:19356662

  10. Spectrally resolved multidepth fluorescence imaging

    PubMed Central

    Luo, Yuan; Zervantonakis, Ioannis K.; Oh, Se Baek; Kamm, Roger D.; Barbastathis, George

    2011-01-01

    We present a multicolor fluorescence imaging modality to visualize in real-time tissue structures emitting multispectral fluorescent light from different focal depths. Each designated spectrum of fluorescent emission from a specific depth within a volumetric tissue is probed by a depth-spectrum selective holographic grating. The grating for each fluorescent color are multiplexed within a volume hologram, which enables simultaneously obtaining multicolored fluorescent information at different depths within a biological tissue sample. We demonstrate the imaging modality's ability to obtain laser-induced multicolored fluorescence images of a biological sample from different depths without scanning. We also experimentally demonstrate that the imaging modality can be simultaneously operated at both fluorescent and bright field modes to provide complementary information of volumetric tissue structures at different depths in real-time. PMID:21950929

  11. Spectrally resolved multidepth fluorescence imaging

    NASA Astrophysics Data System (ADS)

    Luo, Yuan; Zervantonakis, Ioannis K.; Oh, Se Baek; Kamm, Roger D.; Barbastathis, George

    2011-09-01

    We present a multicolor fluorescence imaging modality to visualize in real-time tissue structures emitting multispectral fluorescent light from different focal depths. Each designated spectrum of fluorescent emission from a specific depth within a volumetric tissue is probed by a depth-spectrum selective holographic grating. The grating for each fluorescent color are multiplexed within a volume hologram, which enables simultaneously obtaining multicolored fluorescent information at different depths within a biological tissue sample. We demonstrate the imaging modality's ability to obtain laser-induced multicolored fluorescence images of a biological sample from different depths without scanning. We also experimentally demonstrate that the imaging modality can be simultaneously operated at both fluorescent and bright field modes to provide complementary information of volumetric tissue structures at different depths in real-time.

  12. A novel approach to the discovery of small molecule ligands of CDK2

    PubMed Central

    Martin, Mathew P.; Alam, Riazul; Betzi, Stephane; Ingles, Donna J.; Zhu, Jin-Yi

    2012-01-01

    In an attempt to identify novel small molecule ligands of CDK2 with potential as allosteric inhibitors, we devised a robust and cost-effective fluorescence-based high-throughput screening assay. The assay is based on the specific interaction of CDK2 with the extrinsic fluorophore 8-anilino-1-naphthalene sulfonate (ANS), which binds to a large allosteric pocket adjacent to the ATP site. Hit compounds which displace ANS directly or indirectly from CDK2 are readily classified as ATP site binders or allosteric ligands through the use of staurosporine, which blocks the ATP site without displacing ANS. Pilot screening of 1,453 compounds led to the discovery of 12 compounds with displacement activities (EC50 values) ranging from 6 to 44 μM, all of which were classified as ATP site-directed ligands. Four new Type I inhibitor scaffolds were confirmed by X-ray crystallography. While this small compound library contained only ATP-site directed ligands, the application of this assay to large compound libraries has the potential to reveal previously unrecognized chemical scaffolds suitable for structure-based design of CDK2 inhibitors with new mechanisms of action. PMID:22893598

  13. A new target ligand Ser-Glu for PEPT1-overexpressing cancer imaging.

    PubMed

    Dai, Tongcheng; Li, Na; Zhang, Lingzhi; Zhang, Yuanxing; Liu, Qin

    2016-01-01

    Nanoparticles functionalized with active target ligands have been widely used for tumor-specific diagnosis and therapy. The target ligands include antibodies, peptides, proteins, small molecules, and nucleic acid aptamers. Here, we utilize dipeptide Ser-Glu (DIP) as a new ligand to functionalize polymer-based fluorescent nanoparticles (NPs) for pancreatic cancer target imaging. We demonstrate that in the first step, Ser-Glu-conjugated NPs (NPs-DIP) efficiently bind to AsPC-1 and in the following NPs-DIP are internalized into AsPC-1 in vitro. The peptide transporter 1 inhibition experiment reveals that the targeting effects mainly depend on the specific binding of DIP to peptide transporter 1, which is remarkably upregulated in pancreatic cancer cells compared with varied normal cells. Furthermore, NPs-DIP specifically accumulate in the site of pancreatic tumor xenograft and are further internalized into the tumor cells in vivo after intravenous administration, indicating that DIP successfully enhanced nanoparticles internalization efficacy into tumor cells in vivo. This work establishes Ser-Glu to be a new tumor-targeting ligand and provides a promising tool for future tumor diagnostic or therapeutic applications. PMID:26811678

  14. Sulf1 has ligand-dependent effects on canonical and non-canonical Wnt signalling

    PubMed Central

    Fellgett, Simon W.; Maguire, Richard J.; Pownall, Mary Elizabeth

    2015-01-01

    ABSTRACT Wnt signalling plays essential roles during embryonic development and is known to be mis-regulated in human disease. There are many molecular mechanisms that ensure tight regulation of Wnt activity. One such regulator is the heparan-sulfate-specific 6-O-endosulfatase Sulf1. Sulf1 acts extracellularly to modify the structure of heparan sulfate chains to affect the bio-availability of Wnt ligands. Sulf1 could, therefore, influence the formation of Wnt signalling complexes to modulate the activation of both canonical and non-canonical pathways. In this study, we use well-established assays in Xenopus to investigate the ability of Sulf1 to modify canonical and non-canonical Wnt signalling. In addition, we model the ability of Sulf1 to influence morphogen gradients using fluorescently tagged Wnt ligands in ectodermal explants. We show that Sulf1 overexpression has ligand-specific effects on Wnt signalling: it affects membrane accumulation and extracellular levels of tagged Wnt8a and Wnt11b ligands differently, and inhibits the activity of canonical Wnt8a but enhances the activity of non-canonical Wnt11b. PMID:25681501

  15. A new target ligand Ser–Glu for PEPT1-overexpressing cancer imaging

    PubMed Central

    Dai, Tongcheng; Li, Na; Zhang, Lingzhi; Zhang, Yuanxing; Liu, Qin

    2016-01-01

    Nanoparticles functionalized with active target ligands have been widely used for tumor-specific diagnosis and therapy. The target ligands include antibodies, peptides, proteins, small molecules, and nucleic acid aptamers. Here, we utilize dipeptide Ser–Glu (DIP) as a new ligand to functionalize polymer-based fluorescent nanoparticles (NPs) for pancreatic cancer target imaging. We demonstrate that in the first step, Ser–Glu-conjugated NPs (NPs-DIP) efficiently bind to AsPC-1 and in the following NPs-DIP are internalized into AsPC-1 in vitro. The peptide transporter 1 inhibition experiment reveals that the targeting effects mainly depend on the specific binding of DIP to peptide transporter 1, which is remarkably upregulated in pancreatic cancer cells compared with varied normal cells. Furthermore, NPs-DIP specifically accumulate in the site of pancreatic tumor xenograft and are further internalized into the tumor cells in vivo after intravenous administration, indicating that DIP successfully enhanced nanoparticles internalization efficacy into tumor cells in vivo. This work establishes Ser–Glu to be a new tumor-targeting ligand and provides a promising tool for future tumor diagnostic or therapeutic applications. PMID:26811678

  16. A robust ligand exchange approach for preparing hydrophilic, biocompatible photoluminescent quantum dots

    SciTech Connect

    Wang, Sujuan; Zhou, Changhua; Yuan, Hang; Shen, Huaibin; Zhao, Wenxiu; Ma, Lan; Li, Lin Song

    2013-08-01

    Graphical abstract: - Highlights: • Aqueous CdSe/ZnS QDs were prepared using polymaleic anhydrides as capping ligand. • Effect of reaction temperature and time were systematically studied in the synthesis process. • Water-soluble QDs exhibited a good stability in physiological relevant environment. • The aqueous QDs were applied as biological probe to detect human embryonic stem cell. - Abstract: This paper describes a robust ligand exchange approach for preparing biocompatible CdSe/ZnS quantum dots (QDs) to make bioprobe for effective cell imaging. In this method, polymaleic anhydride (PMA) ligand are first used to replace original hydrophobic ligand (oleic acid) and form a protection shell with multiple hydrophilic groups to coat and protect CdSe/ZnS QDs. The as-prepared aqueous QDs exhibit small particle size, good colloidal stability in aqueous solutions with a wide range of pH, salt concentrations and under thermal treatment, which are necessary for biological applications. The use of this new class of aqueous QDs for effective cell imaging shows strong fluorescence signal to human embryonic stem cell, which demonstrate that PMA coated QDs are fully satisfied with the requirements of preparing high quality biological probe.

  17. Binding of angiogenesis inhibitor kringle 5 to its specific ligands by frontal affinity chromatography.

    PubMed

    Bian, Liujiao; Li, Qian; Ji, Xu

    2015-07-01

    The interactions between angiogenesis inhibitor Kringle 5 and its five specific ligands were investigated by frontal affinity chromatography in combination with fluorescence spectra and site-directed molecular docking. The binding constants of trans-4-(aminomethyl) cyclohexane carboxylic acid (AMCHA), epsilon-aminocaproic acid (EACA), benzylamine, 7-aminoheptanoic acid (7-AHA) and L-lysine to Kringle 5 were 19.0×10(3), 7.97×10(3), 6.45×10(3), 6.07×10(3) and 4.04×10(3) L/mol, respectively. The five ligands bound to Kringle 5 on the lysine binding site in equimolar amounts, which was pushed mainly by hydrogen bond and Van der Waals force. This binding affinity was believed to be dependent on the functional group and flexible feature in ligands. This study will provide an important insight into the binding mechanism of angiogenesis inhibitor Kringle 5 to its specific ligands. PMID:25981289

  18. Divergent Label-free Cell Phenotypic Pharmacology of Ligands at the Overexpressed β2-Adrenergic Receptors

    NASA Astrophysics Data System (ADS)

    Ferrie, Ann M.; Sun, Haiyan; Zaytseva, Natalya; Fang, Ye

    2014-01-01

    We present subclone sensitive cell phenotypic pharmacology of ligands at the β2-adrenergic receptor (β2-AR) stably expressed in HEK-293 cells. The parental cell line was transfected with green fluorescent protein (GFP)-tagged β2-AR. Four stable subclones were established and used to profile a library of sixty-nine AR ligands. Dynamic mass redistribution (DMR) profiling resulted in a pharmacological activity map suggesting that HEK293 endogenously expresses functional Gi-coupled α2-AR and Gs-coupled β2-AR, and the label-free cell phenotypic activity of AR ligands are subclone dependent. Pathway deconvolution revealed that the DMR of epinephrine is originated mostly from the remodeling of actin microfilaments and adhesion complexes, to less extent from the microtubule networks and receptor trafficking, and certain agonists displayed different efficacy towards the cAMP-Epac pathway. We demonstrate that receptor signaling and ligand pharmacology is sensitive to the receptor expression level, and the organization of the receptor and its signaling circuitry.

  19. Acridine-based complex as amino acid anion fluorescent sensor in aqueous solution

    NASA Astrophysics Data System (ADS)

    Dai, Yanpeng; Xu, Kuoxi; Li, Qian; Wang, Chaoyu; Liu, Xiaoyan; Wang, Peng

    2016-03-01

    Novel acridine-based fluorescence sensors containing alaninol ligands, L1 and D1, were designed and synthesized. The structure of the compound was characterized by IR, 1H NMR, 13C NMR, MS spectra. L1 and D1 possess efficient Cu2 + cation ON-OFF selective signaling behavior based on ligand-to-metal binding mechanism at physiological pH condition. Additionally, the L1-Cu(II) and D1-Cu(II) complexes could further serve as reversible OFF-ON signaling sensing ensemble to allow ratiometric response to amino acid anion in aqueous solution.

  20. Sensing Metal Ions with DNA Building Blocks: Fluorescent Pyridobenzimidazole Nucleosides

    PubMed Central

    Kim, Su Jeong; Kool, Eric T.

    2008-01-01

    We describe novel fluorescent N-deoxyribosides (1 and 2) having 2-pyrido-2-benzimidazole and 2-quino-2-benzimidazole as aglycones. The compounds were prepared from the previously unknown heterocyclic precursors and Hoffer’s chlorosugar, yielding alpha anomers as the chief products. X-ray crystal structures confirmed the geometry, and showed that the pyridine and benzimidazole ring systems deviated from coplanarity in the solid state by 154° and 140°, respectively. In methanol the compounds 1 and 2 had absorption maxima at 360 and 370 nm respectively, and emission maxima at 494 and 539 nm. Experiments revealed varied fluorescence responses of the nucleosides to a panel of seventeen monovalent, divalent and trivalent metal ions in methanol. One or both of the nucleosides showed significant changes with ten of the metal ions. The most pronounced spectral changes for ligand-nucleoside 1 included red shifts in fluorescence (Au+, Au3+), strong quenching (Cu2+, Ni2+, Pt2+), and in substantial enhancements in emission intensity coupled with redshifts (Ag+, Cd2+, Zn2+). The greatest spectral changes for ligand-nucleoside 2 included a redshift in fluorescence (Ag+), a blueshift (Cd2+), strong quenching (Pd2+, Pt2+), and in substantial enhancements in emission intensity coupled with a blueshift (Zn2+). The compounds could be readily incorporated into oligodeoxynucleotides, where an initial study revealed that they retained sensitivity to metal ions in aqueous solution, and demonstrated possible cooperative sensing behavior with several ions. The two free nucleosides alone can act as differential sensors for at multiple metal ions, and they are potentially useful monomers for contributing metal ion sensing capability to DNAs. PMID:16669686

  1. The stereochemical diversity of a new SNONS binucleating ligand towards 3d metal ions

    NASA Astrophysics Data System (ADS)

    Naik, Anil D.; Annigeri, Satish M.; Gangadharmath, Umesh B.; Revankar, Vidyanand K.; Mahale, Vinayak B.

    2002-06-01

    A new binucleating ligand containing phenoxide as an endogenous bridging group, 2,6-diformyl- p-cresol bis(2-furanthiocarboxyhydrazone) and its binuclear Co(II), Ni(II), Cu(II) and Zn(II) complexes with chloride ion as an exogenous bridge, have been obtained. The complexes were characterized by elemental analysis, molar conductivities, magnetic moment measurements at room temperature, electronic, IR, 1H-NMR, EPR, FAB spectral studies and thermal data. The copper complex assumes a tetranuclear structure composed of two binuclear units related by a center of symmetry. The dimeric nature of copper(II) complex is supported by FAB. This complex is EPR silent. Room temperature magnetic moment reveals the operation of a significant antiferromagnetic spin exchange between the metal centers. Ligand and its copper and zinc complexes exhibit fluorescence at room temperature in DMF. All the compounds show an appreciable antimicrobial activity.

  2. Modulating protein activity using tethered ligands with mutually exclusive binding sites

    PubMed Central

    Schena, Alberto; Griss, Rudolf; Johnsson, Kai

    2015-01-01

    The possibility to design proteins whose activities can be switched on and off by unrelated effector molecules would enable applications in various research areas, ranging from biosensing to synthetic biology. We describe here a general method to modulate the activity of a protein in response to the concentration of a specific effector. The approach is based on synthetic ligands that possess two mutually exclusive binding sites, one for the protein of interest and one for the effector. Tethering such a ligand to the protein of interest results in an intramolecular ligand–protein interaction that can be disrupted through the presence of the effector. Specifically, we introduce a luciferase controlled by another protein, a human carbonic anhydrase whose activity can be controlled by proteins or small molecules in vitro and on living cells, and novel fluorescent and bioluminescent biosensors. PMID:26198003

  3. Time, the Forgotten Dimension of Ligand Binding Teaching

    ERIC Educational Resources Information Center

    Corzo, Javier

    2006-01-01

    Ligand binding is generally explained in terms of the equilibrium constant K[subscript d] for the protein-ligand complex dissociation. However, both theoretical considerations and experimental data point to the life span of the protein-ligand complex as an important, but generally overlooked, aspect of ligand binding by macromolecules. Short-lived…

  4. Synthesis and characterization of mixed ligand chiral nanoclusters.

    PubMed

    Guven, Zekiye P; Ustbas, Burcin; Harkness, Kellen M; Coskun, Hikmet; Joshi, Chakra P; Besong, Tabot M D; Stellacci, Francesco; Bakr, Osman M; Akbulut, Ozge

    2016-07-28

    Chiral mixed ligand silver nanoclusters were synthesized in the presence of a chiral and an achiral ligand. While the chiral ligand led mostly to the formation of nanoparticles, the presence of the achiral ligand drastically increased the yield of nanoclusters with enhanced chiral properties. PMID:27362744

  5. A novel supramolecular polymer gel constructed by crosslinking pillar[5]arene-based supramolecular polymers through metal-ligand interactions.

    PubMed

    Wang, Pi; Xing, Hao; Xia, Danyu; Ji, Xiaofan

    2015-12-21

    A novel heteroditopic A-B monomer was synthesized and used to construct linear supramolecular polymers utilizing pillar[5]arene-based host-guest interactions. Specifically, upon addition of Cu(2+) ions, the supramolecular polymer chains are crosslinked through metal-ligand interactions, resulting in the formation of a supramolecular polymer gel. Interestingly, this self-organized supramolecular polymer can be used as a novel fluorescent sensor for detecting Cu(2+) ions. PMID:26466511

  6. Single-molecule imaging reveals the stoichiometry change of β2-adrenergic receptors by a pharmacological biased ligand.

    PubMed

    Sun, Yahong; Li, Nan; Zhang, Mingliang; Zhou, Wei; Yuan, Jinghe; Zhao, Rong; Wu, Jimin; Li, Zijian; Zhang, Youyi; Fang, Xiaohong

    2016-06-01

    The stoichiometry of the β2-adrenergic receptor (β2AR) was determined using single-molecule fluorescence imaging in living cells. The results showed that β2AR mainly existed as monomers under physiological conditions and exhibited β-arrestin-dependent dimerization upon stimulation with the pharmacological biased ligand carvedilol. The association of β2AR dimerization with biased signalling is revealed. PMID:27090587

  7. Two Ce-containing 3D metal–organic frameworks: In situ formation of ligand (DDPD)

    SciTech Connect

    Cao, Xinyu; Yu, Liqiong; Huang, Rudan

    2014-02-15

    Hydrothermal reactions of cerium nitrate and 5-hydroxyisophthalic acid (OH-H{sub 2}BDC) produce two new metal–organic frameworks (MOFs), ([Ce(DDPD){sub 1.5}(H{sub 2}O){sub 2.5}]·4H{sub 2}O){sub n} (1) and ([Ce(OH-BDC)(OH-HBDC)(H{sub 2}O){sub 2}]·2H{sub 2}O]{sub n} (2) (DDPD(II)=5,10-dioxo-5,10-dihydro-4,9-dioxapyrene-2,7-dicarboxylate(II)). These two complexes have been characterized by elemental analysis, IR, TG, and single-crystal X-ray diffraction. It was remarkable that the in situ reaction of OH-H{sub 2}BDC to DDPD(II) was found in complex 1. In 1, Ce(III) ions are bridged by DDPD ligands to form infinite 1D chain, which is further connected via DDPD ligands to form 3D structure. Complex 2 possesses a neutral noninterpenetrating 2D layer structure. Furthermore, the fluorescence properties and magnetic behavior of 1 and 2 have been investigated. - Graphical abstract: In complex 1, the in situ reaction of OH-H{sub 2}BDC to DDPD(II) was found. Complex 1 features a 3D network structure. Adjacent Ce(III) ions are bridged by two carboxylate groups to form a 1D infinite inorganic chain, and further linked by the DDPD(II) ligands. Display Omitted - Highlights: • Complexes 1 and 2 was synthesized via hydrothermal methods. • In situ reaction of OH-H{sub 2}BDC to DDPD(II) was found in complex 1. • Ce(III) ions are bridged by the DDPD(II) ligands to generate a 3D structure in complex 1. • Complex 2 possesses a neutral noninterpenetrating 2D layer structure. • Fluorescent properties and magnetic behavior of 1 and 2 have been studied.

  8. Zinc complexes exhibiting highly efficient thermally activated delayed fluorescence and their application to organic light-emitting diodes.

    PubMed

    Sakai, Yumi; Sagara, Yuta; Nomura, Hiroko; Nakamura, Nozomi; Suzuki, Yoshitake; Miyazaki, Hiroshi; Adachi, Chihaya

    2015-02-21

    Metal complexes emitting thermally activated delayed fluorescence based on intra-ligand charge transfer and enhanced by metallization were synthesized. Organic light-emitting diodes using a thermally stable zinc complex processed by vacuum vapor deposition achieved an external quantum efficiency of nearly 20%. PMID:25605520

  9. A Novel Chromone Schiff-Base Fluorescent Chemosensor for Cd(II) Based on C=N Isomerization.

    PubMed

    Yan, Jun; Fan, Long; Qin, Jing-Can; Li, Chao-Rui; Yang, Zheng-Yin

    2016-05-01

    A new chromone Schiff-base fluorescent probe 7'-methoxychromone-3'-methylidene-1,2,4-triazole-3-imine (L) was designed and synthesized for selective recognition Cd(2+). With the fluorescence titration and the ESI-MS data, we reach the conclusion that the binding mode of the ligand-metal (L-Cd (2+) ) complex is 1:1. The sensor showed a strong fluorescence enhancement in ethanol system of Cd(2+) (excitation 409 nm and emission 462 nm) and the sensing mechanism based on the fact that C=N isomerization can be used to explain this phenomenon. PMID:27048223

  10. Fluorescence enhancement of europium(III) perchlorate by benzoic acid on bis(benzylsulfinyl)methane complex and its binding characteristics with the bovine serum albumin (BSA)

    NASA Astrophysics Data System (ADS)

    Zhang, Jing; Li, Wen-Xian; Ao, Bo-Yang; Feng, Shu-Yan; Xin, Xiao-Dong

    2014-01-01

    A novel ligand with double sulfinyl groups, bis(benzylsulfinyl)methane L, was synthesized by a new method. Its novel ternary complex, EuL2.5ṡL‧·(ClO4)2ṡ5H2O, has been synthesized [using L as the first ligand, and benzoic acid L‧ as the second ligand], and characterized by elemental analysis, molar conductivity, coordination titration analysis, FTIR, TG-DSC, 1H NMR and UV-vis. In order to study the effect of the second ligand on the fluorescence properties of rare-earth sulfoxide complex, a novel binary complex EuL2.5·(ClO4)3·3H2O has been synthesized. Photoluminescent measurement showed that the first ligand L could efficiently transfer the energy to Eu3+ ions in the complex. Furthermore, the detailed luminescence analyses on the rare earth complexes indicated that the ternary Eu (III) complex manifested stronger fluorescence intensities, longer lifetimes, and higher fluorescence quantum efficiencies than the binary Eu (III) materials. After introducing the second ligand L‧, the fluorescence emission intensities and fluorescence lifetimes of the ternary complex enhanced more obviously than the binary complex. This illustrated that the presence of both the first ligand L and the second ligand L‧ could sensitize fluorescence intensities of Eu (III) ions. The fluorescence spectra, fluorescence lifetime and phosphorescence spectra were also discussed. To explore the potential biological value of Eu (III) complexes, the binding interaction among Eu (III) complexes and bovine serum albumin (BSA) was studied by fluorescence spectrum. The result indicated that the reaction between Eu (III) complexes and BSA was a static quenching procedure. The binding site number, n, of 0.60 and 0.78, and binding constant, Ka, of 0.499 and 4.46 were calculated according to the double logarithm regression equation, respectively for EuL2.5ṡL‧ṡ(ClO4)2ṡ5H2O and EuL2.5ṡ(ClO4)3ṡ3H2O systems.

  11. An adaptable pentaloop defines a robust neomycin-B RNA aptamer with conditional ligand-bound structures

    PubMed Central

    Ilgu, Muslum; Fulton, D. Bruce; Yennamalli, Ragothaman M.; Lamm, Monica H.; Sen, Taner Z.; Nilsen-Hamilton, Marit

    2014-01-01

    Aptamers can be highly specific for their targets, which implies precise molecular recognition between aptamer and target. However, as small polymers, their structures are more subject to environmental conditions than the more constrained longer RNAs such as those that constitute the ribosome. To understand the balance between structural and environmental factors in establishing ligand specificity of aptamers, we examined the RNA aptamer (NEO1A) previously reported as specific for neomycin-B. We show that NEO1A can recognize other aminoglycosides with similar affinities as for neomycin-B and its aminoglycoside specificity is strongly influenced by ionic strength and buffer composition. NMR and 2-aminopurine (2AP) fluorescence studies of the aptamer identified a flexible pentaloop and a stable binding pocket. Consistent with a well-structured binding pocket, docking analysis results correlated with experimental measures of the binding energy for most ligands. Steady state fluorescence studies of 2AP-substituted aptamers confirmed that A16 moves to a more solvent accessible position upon ligand binding while A14 moves to a less solvent accessible position, which is most likely a base stack. Analysis of binding affinities of NEO1A sequence variants showed that the base in position 16 interacts differently with each ligand and the interaction is a function of the buffer constituents. Our results show that the pentaloop provides NEO1A with the ability to adapt to external influences on its structure, with the critical base at position 16 adjusting to incorporate each ligand into a stable pocket by hydrophobic interactions and/or hydrogen bonds depending on the ligand and the ionic environment. PMID:24757168

  12. Fluorescent microthermographic imaging

    SciTech Connect

    Barton, D.L.

    1993-09-01

    In the early days of microelectronics, design rules and feature sizes were large enough that sub-micron spatial resolution was not needed. Infrared or IR thermal techniques were available that calculated the object`s temperature from infrared emission. There is a fundamental spatial resolution limitation dependent on the wavelengths of light being used in the image formation process. As the integrated circuit feature sizes began to shrink toward the one micron level, the limitations imposed on IR thermal systems became more pronounced. Something else was needed to overcome this limitation. Liquid crystals have been used with great success, but they lack the temperature measurement capabilities of other techniques. The fluorescent microthermographic imaging technique (FMI) was developed to meet this need. This technique offers better than 0.01{degrees}C temperature resolution and is diffraction limited to 0.3 {mu}m spatial resolution. While the temperature resolution is comparable to that available on IR systems, the spatial resolution is much better. The FMI technique provides better spatial resolution by using a temperature dependent fluorescent film that emits light at 612 nm instead of the 1.5 {mu}m to 12 {mu}m range used by IR techniques. This tutorial starts with a review of blackbody radiation physics, the process by which all heated objects emit radiation to their surroundings, in order to understand the sources of information that are available to characterize an object`s surface temperature. The processes used in infrared thermal imaging are then detailed to point out the limitations of the technique but also to contrast it with the FMI process. The FMI technique is then described in detail, starting with the fluorescent film physics and ending with a series of examples of past applications of FMI.

  13. Fluorescence dynamics of green fluorescent protein in AOT reversed micelles.

    PubMed

    Uskova, M A; Borst, J W; Hink, M A; van Hoek, A; Schots, A; Klyachko, N L; Visser, A J

    2000-09-15

    We have used the enhanced green fluorescent protein (EGFP) to investigate the properties of surfactant-entrapped water pools in organic solvents (reversed micelles) with steady-state and time-resolved fluorescence methods. The surfactant used was sodium bis(2-ethylhexyl)sulfosuccinate (AOT) and the organic solvents were isooctane and (the more viscous) dodecane, respectively. The water content of the water pools could be controlled through the parameter w0, which is the water-to-surfactant molar ratio. With steady-state fluorescence, it was observed that subtle fluorescence changes could be noted in reversed micelles of different water contents. EGFP can be used as a pH-indicator of the water droplets in reversed micelles. Time-resolved fluorescence methods also revealed subtle changes in fluorescence decay times when the results in bulk water were compared with those in reversed micelles. The average fluorescence lifetimes of EGFP scaled with the relative fluorescence intensities. Time-resolved fluorescence anisotropy of EGFP in aqueous solution and reversed micelles yielded single rotational correlation times. Geometrical considerations could assign the observed correlation times to dehydrated protein at low w0 and internal EGFP rotation within the droplet at the highest w0. PMID:11036971

  14. Emerging fluorescent protein technologies.

    PubMed

    Enterina, Jhon Ralph; Wu, Lanshi; Campbell, Robert E

    2015-08-01

    Fluorescent proteins (FPs), such as the Aequorea jellyfish green FP (GFP), are firmly established as fundamental tools that enable a wide variety of biological studies. Specifically, FPs can serve as versatile genetically encoded markers for tracking proteins, organelles, or whole cells, and as the basis for construction of biosensors that can be used to visualize a growing array of biochemical events in cells and tissues. In this review we will focus on emerging applications of FPs that represent unprecedented new directions for the field. These emerging applications include new strategies for using FPs in biosensing applications, and innovative ways of using FPs to manipulate protein function or gene expression. PMID:26043278

  15. Fluorescent penetrant inspection

    NASA Technical Reports Server (NTRS)

    Sastri, Sankar

    1990-01-01

    The purpose of this experiment is to familiarize the student with fluorescent penetrant inspection and to relate it to classification of various defects. The penetrant method of nondestructive testing is a method for finding discontinuities open to the surface in solids and essentially nonporous bodies. The method employs a penetrating liquid which is applied over the surface and enters the discontinuity or crack. After the excess of penetrant has been cleaned from the surface, the penetrant which exudes or is drawn back out of the crack indicates the presence and location of a discontinuity. The experimental procedure is described.

  16. Fluorescence analyzer for lignin

    DOEpatents

    Berthold, John W.; Malito, Michael L.; Jeffers, Larry

    1993-01-01

    A method and apparatus for measuring lignin concentration in a sample of wood pulp or black liquor comprises a light emitting arrangement for emitting an excitation light through optical fiber bundles into a probe which has an undiluted sensing end facing the sample. The excitation light causes the lignin concentration to produce fluorescent emission light which is then conveyed through the probe to analyzing equipment which measures the intensity of the emission light. Measures a This invention was made with Government support under Contract Number DOE: DE-FC05-90CE40905 awarded by the Department of Energy (DOE). The Government has certain rights in this invention.

  17. Fluorescent temperature sensor

    SciTech Connect

    Baker, Gary A; Baker, Sheila N; McCleskey, T Mark

    2009-03-03

    The present invention is a fluorescent temperature sensor or optical thermometer. The sensor includes a solution of 1,3-bis(1-pyrenyl)propane within a 1-butyl-1-1-methylpyrrolidinium bis(trifluoromethylsulfonyl)imide ionic liquid solvent. The 1,3-bis(1-pyrenyl)propane remains unassociated when in the ground state while in solution. When subjected to UV light, an excited state is produced that exists in equilibrium with an excimer. The position of the equilibrium between the two excited states is temperature dependent.

  18. Fluorescence Detection In Electrophoresis

    NASA Astrophysics Data System (ADS)

    Swarner, Susan

    1988-04-01

    Fluorescence detection is in common usage in forensic science laboratories for the visualization of three enzyme markers. The fluorogenic substrates, 4-methylumbelliferyl phosphate, 4-methylutbel-liveryl acetate, and fluorecein diacetate, are acted upon by the enzymes Erythrocyte Acid Phospha, tase, Esterase-D, and Carbonic Anhydrase-III, respectively, to produce compounds visible to the analyst when viewed with transmitted UV light at 365 nm. Additionally, the choice of fluorogenic corn, pounds may help detect a specific enzyme from a related enzyme. One of the responsibilities of a forensic science laboratory may be the analysis of blood for genetically controlled polymorphic enzymes and protein markers. The genetic markers are said to be polymorphic because each exhibits types which can be differentiated and allows for the inclusion or exclusion of possible-donors of the blood. Each genetic marker can be separated into these recognizable types by electrophoresis, a technique which separates compounds based on electrical charges. Electrophoresis is conducted by placing a portion or extract of each bloodstain into a support medium which will conduct electricity. This is known as a plate or membrane. By controlling the pH of the buffer and the potential that is applied to the plate, the analyst can achieve separation of the types within an enzyme marker. The types appear as differing patterns of bands. Once the bloodstain has been subjected to electrophoresis, the enzymes must be visualized. This is generally best accomplished by using the specific activity of the enzyme. For the enzymes described in the present work, the visualization is performed by over-layering the plate with a piece of filter paper that 'has been saturated with the appropriate non-fluorescent substrate and buffer. The bands of enzyme, which is now in discrete patterns, will act upon the non-fluorescent substrate to create a fluorescent compound. The plate is then viewed with transmitted UV

  19. A cationic copper(I) iodide cluster MOF exhibiting unusual ligand assisted thermochromism.

    PubMed

    Yadav, Ashok; Srivastava, Anant Kumar; Balamurugan, Ayyakkalai; Boomishankar, Ramamoorthy

    2014-06-14

    By employing a tridentate thiophosphoramide ligand, [(NHAQ)3P[double bond, length as m-dash]S] (AQ = 3-quinolinyl), a cationic MOF, {[Cu6I5(L(1))2](OH)·3DMF·2.5MeOH}n, was synthesized. Photo-physical studies on the 2D-MOF showed an unusual thermochromic behaviour emitting a blue fluorescence at 298 K due to the AQ chromophore and an orange-yellow phosphorescence at 77 K due to the [Cu6I5](+) unit. PMID:24760039

  20. Sliding tethered ligands add topological interactions to the toolbox of ligand-receptor design

    NASA Astrophysics Data System (ADS)

    Bauer, Martin; Kékicheff, Patrick; Iss, Jean; Fajolles, Christophe; Charitat, Thierry; Daillant, Jean; Marques, Carlos M.

    2015-09-01

    Adhesion in the biological realm is mediated by specific lock-and-key interactions between ligand-receptor pairs. These complementary moieties are ubiquitously anchored to substrates by tethers that control the interaction range and the mobility of the ligands and receptors, thus tuning the kinetics and strength of the binding events. Here we add sliding anchoring to the toolbox of ligand-receptor design by developing a family of tethered ligands for which the spacer can slide at the anchoring point. Our results show that this additional sliding degree of freedom changes the nature of the adhesive contact by extending the spatial range over which binding may sustain a significant force. By introducing sliding tethered ligands with self-regulating length, this work paves the way for the development of versatile and reusable bio-adhesive substrates with potential applications for drug delivery and tissue engineering.

  1. A General Ligand Design for Gold Catalysis allowing Ligand-Directed Anti Nucleophilic Attack of Alkynes

    PubMed Central

    Wang, Yanzhao; Wang, Zhixun; Li, Yuxue; Wu, Gongde; Cao, Zheng; Zhang, Liming

    2014-01-01

    Most homogenous gold catalyses demand ≥0.5 mol % catalyst loading. Due to the high cost of gold, these reactions are unlikely to be applicable in medium or large scale applications. Here we disclose a novel ligand design based on the privileged biphenyl-2-phosphine framework that offers a potentially general approach to dramatically lowering catalyst loading. In this design, an amide group at the 3’ position of the ligand framework directs and promotes nucleophilic attack at the ligand gold complex-activated alkyne, which is unprecedented in homogeneous gold catalysis considering the spatial challenge of using ligand to reach antiapproaching nucleophile in a linear P-Au-alkyne centroid structure. With such a ligand, the gold(I) complex becomes highly efficient in catalyzing acid addition to alkynes, with a turnover number up to 99,000. Density functional theory calculations support the role of the amide moiety in directing the attack of carboxylic acid via hydrogen bonding. PMID:24704803

  2. Controlling Gold Nanoclusters by Diphospine Ligands

    SciTech Connect

    Chen, Jing; Zhang, Qianfan; Bonaccorso, Timary A.; Williard, Paul G.; Wang, Lai S.

    2014-01-08

    We report the synthesis and structure determination of a new Au22 nanocluster coordinated by six bidentate diphosphine ligands: 1,8-bis(diphenylphosphino) octane (L8 for short). Single crystal x-ray crystallography and electrospray ionization mass spectrometry show that the cluster assembly is neutral and can be formulated as Au22(L8)6. The Au22 core consists of two Au11 units clipped together by four L8 ligands, while the additional two ligands coordinate to each Au11 unit in a bidentate fashion. Eight gold atoms at the interface of the two Au11 units are not coordinated by any ligands. Four short gold-gold distances (2.64?2.65 Å) are observed at the interface of the two Au11 clusters as a result of the clamping force of the four clipping ligands and strong electronic interactions. The eight uncoordinated surface gold atoms in the Au22(L8)6 nanocluster are unprecedented in atom-precise gold nanoparticles and can be considered as potential in-situ active sites for catalysis.

  3. Ligand-responsive RNA mechanical switches.

    PubMed

    Boerneke, Mark A; Hermann, Thomas

    2015-01-01

    Ligand-responsive RNA mechanical switches represent a new class of simple switching modules that adopt well-defined ligand-free and bound conformational states, distinguishing them from metabolite-sensing riboswitches. Initially discovered in the internal ribosome entry site (IRES) of hepatitis C virus (HCV), these RNA switch motifs were found in the genome of diverse other viruses. Although large variations are seen in sequence and local secondary structure of the switches, their function in viral translation initiation that requires selective ligand recognition is conserved. We recently determined the crystal structure of an RNA switch from Seneca Valley virus (SVV) which is able to functionally replace the switch of HCV. The switches from both viruses recognize identical cognate ligands despite their sequence dissimilarity. Here, we describe the discovery of 7 new switches in addition to the previously established 5 examples. We highlight structural and functional features unique to this class of ligand-responsive RNA mechanical switches and discuss implications for therapeutic development and the construction of RNA nanostructures. PMID:26158858

  4. Dockomatic - automated ligand creation and docking

    PubMed Central

    2010-01-01

    Background The application of computational modeling to rationally design drugs and characterize macro biomolecular receptors has proven increasingly useful due to the accessibility of computing clusters and clouds. AutoDock is a well-known and powerful software program used to model ligand to receptor binding interactions. In its current version, AutoDock requires significant amounts of user time to setup and run jobs, and collect results. This paper presents DockoMatic, a user friendly Graphical User Interface (GUI) application that eases and automates the creation and management of AutoDock jobs for high throughput screening of ligand to receptor interactions. Results DockoMatic allows the user to invoke and manage AutoDock jobs on a single computer or cluster, including jobs for evaluating secondary ligand interactions. It also automates the process of collecting, summarizing, and viewing results. In addition, DockoMatic automates creation of peptide ligand .pdb files from strings of single-letter amino acid abbreviations. Conclusions DockoMatic significantly reduces the complexity of managing multiple AutoDock jobs by facilitating ligand and AutoDock job creation and management. PMID:21059259

  5. Determining ligand specificity of Ly49 receptors.

    PubMed

    Lavender, Kerry J; Kane, Kevin P

    2010-01-01

    Ly49 receptors in rodents, like KIR in humans, play an integral role in the regulation of NK cell activity. Some inhibitory Ly49 are known to interact with specific MHC I alleles to maintain tolerance to self tissues, and NK activation is triggered upon the loss of inhibitory signals due to pathological downregulation of self MHC I. Although a virally encoded ligand has been identified that can trigger NK cytotoxicity through an activating Ly49, some activating Ly49 also recognize MHC I and the role of most activating receptors in NK effector function remains poorly defined. As many Ly49 remain orphan receptors, we describe methods to unambiguously discern receptor-ligand pairs. Additionally, we describe a method for the mutagenesis of Ly49 and MHC ligands that can be used to define the motifs conferring receptor specificity for their ligands. Further elucidation of Ly49 ligands is required to continue to define the role of Ly49 in regulating NK cell effector function and may give vital clues to the role of KIR in human health and disease. PMID:20033649

  6. Development of a fluorescent cryocooler

    SciTech Connect

    Edwards, B.C.; Buchwald, M.I.; Epstein, R.I.; Gosnell, T.R.; Mungan, C.E.

    1995-10-01

    Recent work at Los Alamos National Laboratory has demonstrated the physical principles for a new type of solid-state cryocooler based on anti-Stokes fluorescence. Design studies indicate that a vibration-free, low-mass ``fluorescent cryocooler`` could operate for years with efficiencies and cooling powers comparable to current commercial systems. This paper presents concepts for a fluorescent cryocooler, design considerations and expected performance.

  7. Fluorescence quenching of water-soluble conjugated polymer by metal cations and its application in sensor

    NASA Astrophysics Data System (ADS)

    Chen, Yan-Guo; Zhao, Dan; He, Zhi-Ke; Ai, Xin-Ping

    2007-02-01

    The effects of different metal cations on the fluorescence of water-soluble conjugated polymer (CP) and their quenching mechanism have been explored. Most transition metal cations, especially noble metal cations, such as Pd 2+, Ru 3+, and Pt 2+ possessed higher quenching efficiency to CP fluorescence than that of the main group metal cations and other transition metal cations, which have filled or half-full outmost electron layer configurations. Base on this, rapid, sensitive detection of noble metal cations can be realized and a novel quencher-tether-ligand (QTL) probe was developed to detect avidin and streptavidin.

  8. Fluorescence-Based Sensors

    NASA Astrophysics Data System (ADS)

    Orellana, Guillermo

    The natural luminescent phenomena (from the Latin words "lumen" and "essentia", i.e., "made of light") such as northern lights (aurora borealis), marine brightness, glow-worms, shining putrid fish scales, "bluish"- appearing water when contained in certain wooden cups (quinine fluorescence), some stones heated at high temperatures with reducing agents (BaS phosphorescence), or light emitted while crushing sugar (triboluminescence) already fascinated our ancestors. Nowadays we understand that ultraviolet and visible emission of light originates from a competitive deactivation pathway of the lowest electronic excited state of atoms and molecules that produces the so called luminescence (the sub-terms fluorescence and phosphorescence just designate whether the return of the excited to the ground state is an "allowed" or "forbidden" process, namely it is fast or slow, the loosely-defined border between them being a 1-μs-1 rate constant). Actually, luminescence is the only method to generate light in the known Universe regardless it is powered by the nuclear reactions in the stars, the ohmical heating in bulbs, an electric discharge, the absorption of light or a (bio)chemical reaction (chemiluminescence).

  9. Ligand identification using electron-density map correlations

    SciTech Connect

    Terwilliger, Thomas C.; Adams, Paul D.; Moriarty, Nigel W.; Cohn, Judith D.

    2007-01-01

    An automated ligand-fitting procedure is applied to (F{sub o} − F{sub c})exp(iϕ{sub c}) difference density for 200 commonly found ligands from macromolecular structures in the Protein Data Bank to identify ligands from density maps. A procedure for the identification of ligands bound in crystal structures of macromolecules is described. Two characteristics of the density corresponding to a ligand are used in the identification procedure. One is the correlation of the ligand density with each of a set of test ligands after optimization of the fit of that ligand to the density. The other is the correlation of a fingerprint of the density with the fingerprint of model density for each possible ligand. The fingerprints consist of an ordered list of correlations of each the test ligands with the density. The two characteristics are scored using a Z-score approach in which the correlations are normalized to the mean and standard deviation of correlations found for a variety of mismatched ligand-density pairs, so that the Z scores are related to the probability of observing a particular value of the correlation by chance. The procedure was tested with a set of 200 of the most commonly found ligands in the Protein Data Bank, collectively representing 57% of all ligands in the Protein Data Bank. Using a combination of these two characteristics of ligand density, ranked lists of ligand identifications were made for representative (F{sub o} − F{sub c})exp(iϕ{sub c}) difference density from entries in the Protein Data Bank. In 48% of the 200 cases, the correct ligand was at the top of the ranked list of ligands. This approach may be useful in identification of unknown ligands in new macromolecular structures as well as in the identification of which ligands in a mixture have bound to a macromolecule.

  10. Miniature fiber optic sensor based on fluorescence energy transfer

    NASA Astrophysics Data System (ADS)

    Meadows, David L.; Schultz, Jerome S.

    1992-04-01

    Optical fiber biosensors based on fluorescence assays have several distinct advantages when measuring biological analytes such as metabolites, cofactors, toxins, etc. Not only are optical signals immune to electronic interferences, but the polychromatic nature of most fluorochemical assays provides more potentially useful data about the system being studied. One of the most common difficulties normally encountered with optical biosensors is the inability to routinely recalibrate the optical and electronic components of the system throughout the life of the sensor. With this in mind, we present an optical fiber assay system for glucose based on a homogeneous singlet/singlet energy transfer assay along with the electronic instrumentation built to support the sensor system. In the sensor probe, glucose concentrations are indirectly measured from the level of fluorescence quenching caused by the homogeneous competition assay between TRITC labeled concanavalin A (receptor) and FITC labeled Dextran (ligand). The FITC signal is used to indicate glucose concentrations and the TRITC signal is used for internal calibration. Data is also presented on a protein derivatization procedure that was used to prevent aggregation of the receptor protein in solution. Also, a molecular model is described for the singlet/singlet energy transfer interactions that can occur in a model system composed of a monovalent ligand (FITC labeled papain) and a monovalent receptor (TRITC labeled concanavalin A).

  11. Novel Fe (III) heterochelates: Synthesis, structural features and fluorescence studies

    NASA Astrophysics Data System (ADS)

    Modi, C. K.; Jani, D. H.; Patel, H. S.; Pandya, H. M.

    2010-04-01

    Fluorescence properties of five 4-acyl pyrazolone based hydrazides (H 2SB n) and their Fe (III) heterochelates of the type [Fe(SB n)(L)(H 2O)]· mH 2O [H 2SB n = nicotinic acid [1-(3-methyl-5-oxo-1-phenyl-4,5-di hydro-1H-pyrazol-4yl)-acylidene]-hydrazide; where acyl = -CH 3, m = 4 ( H2SB1); -C 6H 5, m = 2 ( H2SB2); -CH 2-CH 3, m = 3 ( H2SB3); -CH 2-CH 2-CH 3, m = 1.5 ( H2SB4); -CH 2-C 6H 5, m = 1.5 ( H2SB5) and HL = 1-cyclopropyl-6-fluoro-4-oxo-7-(piperazin-1-yl)-1,4-dihydroquinoline-3-carboxylic acid] were studied at room temperature. The fluorescence spectra of heterochelates show red shift, which may be due to the chelation by the ligands to the metal ion. It enhances ligand ability to accept electrons and decreases the electron transition energy. The kinetic parameters such as order of reaction ( n), energy of activation ( Ea), entropy ( S*), pre-exponential factor ( A), enthalpy ( H*) and Gibbs free energy ( G*) have been reported.

  12. Reversible fluorescence photoswitching in DNA.

    PubMed

    Smith, Darren A; Holliger, Philipp; Flors, Cristina

    2012-08-30

    We describe the engineering of reversible fluorescence photoswitching in DNA with high-density substitution, and its applications in advanced fluorescence microscopy methods. High-density labeling of DNA with cyanine dyes can be achieved by polymerase chain reaction using a modified DNA polymerase that has been evolved to efficiently incorporate Cy3- and Cy5-labeled cytosine base analogues into double-stranded DNA. The resulting biopolymer, "CyDNA", displays hundreds of fluorophores per DNA strand and is strongly colored and highly fluorescent, although previous observations suggest that fluorescence quenching at such high density might be a concern, especially for Cy5. Herein, we first investigate the mechanisms of fluorescence quenching in CyDNA and we suggest that two different mechanisms, aggregate formation and resonance energy transfer, are responsible for fluorescence quenching at high labeling densities. Moreover, we have been able to re-engineer CyDNA into a reversible fluorescence photoswitchable biopolymer by using the properties of the Cy3-Cy5 pair. This novel biopolymer constitutes a new class of photoactive DNA-based nanomaterial and is of great interest for advanced microscopy applications. We show that reversible fluorescence photoswitching in CyDNA can be exploited in optical lock-in detection imaging. It also lays the foundations for improved and sequence-specific super-resolution fluorescence microscopy of DNA. PMID:22861666

  13. Characteristic molecular vibrations of adenosine receptor ligands.

    PubMed

    Chee, Hyun Keun; Yang, Jin-San; Joung, Je-Gun; Zhang, Byoung-Tak; Oh, S June

    2015-02-13

    Although the regulation of membrane receptor activation is known to be crucial for molecular signal transduction, the molecular mechanism underlying receptor activation is not fully elucidated. Here we study the physicochemical nature of membrane receptor behavior by investigating the characteristic molecular vibrations of receptor ligands using computational chemistry and informatics methods. By using information gain, t-tests, and support vector machines, we have identified highly informative features of adenosine receptor (AdoR) ligand and corresponding functional amino acid residues such as Asn (6.55) of AdoR that has informative significance and is indispensable for ligand recognition of AdoRs. These findings may provide new perspectives and insights into the fundamental mechanism of class A G protein-coupled receptor activation. PMID:25622891

  14. Lanthanide clusters with azide capping ligands.

    PubMed

    Moore, Brian F; Emge, Thomas J; Brennan, John G

    2013-05-20

    Weakly binding azide ligands have been used as surface caps in the synthesis of lanthanide oxo and selenido clusters. Addition of NaN3 and Na2O to in situ prepared solutions of Ln(SePh)3 in pyridine results in the formation of (py)18Sm6Na2O2(N3)16 or (py)10Ln6O2(N3)12(SePh)2 (Ln = Ho, Er), with the Sm and Er compounds characterized by low temperature single crystal X-ray diffraction. Attempts to prepare chalcogenido derivatives by ligand-based redox reactions using elemental Se were successful in the preparation of (py)10Er6O2(SeSe)2(N3)10, a diselenido cluster having crystallographic disorder due to some site sharing of both SeSe and N3 ligands. These compounds all detonate when heated. PMID:23639142

  15. Enhancing the Photostability of Arylvinylenebipyridyl Compounds as Fluorescent Indicators for Intracellular Zinc(II) Ions.

    PubMed

    Yuan, Zhao; Younes, Ali H; Allen, John R; Davidson, Michael W; Zhu, Lei

    2015-06-01

    Arylvinylenebipyridyl (AVB) ligands are bright, zinc(II)-sensitive fluoroionophores. The applicability of AVBs as fluorescent indicators for imaging cellular zinc(II), however, is limited by low photostability, partially attributable to the photoisomerization of the vinylene functionality. Two configurationally immobilized (i.e., "locked") AVB analogues are prepared in this work. The zinc(II)-sensitive photophysical properties and zinc(II) affinities of both AVBs and their locked analogues are characterized in organic and aqueous media. The zinc(II) sensitivity of the emission is attributed to the zinc(II)-dependent energies of the charge transfer excited states of these compounds. The configurationally locked ligands have improved photostability, while maintaining the brightness and zinc(II) sensibility of their AVB progenitors. The feasibility of the "locked" AVB analogues with improved photostability for imaging intracellular Zn(II) of eukaryotic cells using laser confocal fluorescence microscopy is demonstrated. PMID:25942357

  16. A dinuclear cadmium(II) Schiff base thiocyanato complex: crystal structure and fluorescence.

    PubMed

    Shit, Shyamapada; Sankolli, Ravish; Guru Row, Tayur N

    2014-01-01

    A new dinuclear cadmium(II) complex, [Cd(L)(NCS)]2 (1) has been synthesized using a potentially tetradentate Schiff base ligand HL, 2-((E)-(2-(diethylamino)ethylimino)methyl)-6-methoxyphenol, obtained by the condensation of 2-diethylaminoethylamine and o-vanillin, and characterized by different physicochemical techniques. Crystal structure of the title complex was unambiguously established by single crystal X-ray diffraction which reveals that metal centers are connected by bridging phenolato and chelating methoxy oxygen atoms of the coordinating Schiff bases and embedded in severely distorted octahedral geometries. Fluorescence properties of the ligand and its complex, studied at room temperature indicate that later may serve as strong fluorescent emitter. PMID:24664327

  17. Colocalization of Estrogen Receptors with the Fluorescent Tamoxifen Derivative, FLTX1, Analyzed by Confocal Microscopy.

    PubMed

    Morales, Araceli; Marín, Raquel; Marrero-Alonso, Jorge; Boto, Alicia; Díaz, Mario

    2016-01-01

    Tamoxifen is a selective estrogen receptor modulator that competitively binds the ligand-binding domain of estrogen receptors. Binding of tamoxifen displaces its cognate ligand, 17β-estradiol, thereby hampering the activation of estrogen receptors. Cellular labeling of ER is typically carried out using specific antibodies which require permeabilization of cells, incubation with secondary antibodies, and are expensive and time consuming. In this article, we describe the usefulness of FLTX1, a novel fluorescent tamoxifen derivative, which allows the labeling of estrogen receptors in immunocytochemistry and immunohistochemistry studies, both under permeabilized and non-permeabilized conditions. Further, besides labeling canonical estrogen receptors, this novel fluorescent probe is also suitable for the identification of unconventional targets such membrane estrogen receptors as well as other noncanonical targets, some of which are likely responsible for the number of undesired side effects reported during long-term tamoxifen treatments. PMID:26585134

  18. Synthesis, fluorescence study and biological evaluation of three Zn(II) complexes with Paeonol Schiff base.

    PubMed

    Qin, Dong-dong; Yang, Zheng-yin; Qi, Gao-fei

    2009-10-01

    The synthesis of three Paeonol Schiff base ligand and their Zn(II) complexes are reported. The complexes were fully characterized by IR, (1)H NMR, elemental analysis and molar conductivity. The experiment results show the three Zn(II) complexes can emit bright fluorescence at room temperature in DMF solution and solid state. The fluorescence quantum yields (Phi) of three Schiff base ligands and their Zn(II) complexes were calculated using quinine sulfate as the reference with a known Phi(R) of 0.546 in 1.0N sulfuric acid. Furthermore, in order to develop these Zn(II) complexes' biological value, the antioxidant activities against hydroxyl radicals (OH*) were evaluated. The results show the three complexes possess excellent ability to scavenge hydroxyl radicals. PMID:19632146

  19. Synthesis, fluorescence study and biological evaluation of three Zn(II) complexes with Paeonol Schiff base

    NASA Astrophysics Data System (ADS)

    Qin, Dong-dong; Yang, Zheng-yin; Qi, Gao-fei

    2009-10-01

    The synthesis of three Paeonol Schiff base ligand and their Zn(II) complexes are reported. The complexes were fully characterized by IR, 1H NMR, elemental analysis and molar conductivity. The experiment results show the three Zn(II) complexes can emit bright fluorescence at room temperature in DMF solution and solid state. The fluorescence quantum yields ( Φ) of three Schiff base ligands and their Zn(II) complexes were calculated using quinine sulfate as the reference with a known ΦR of 0.546 in 1.0N sulfuric acid. Furthermore, in order to develop these Zn(II) complexes' biological value, the antioxidant activities against hydroxyl radicals (OH rad ) were evaluated. The results show the three complexes possess excellent ability to scavenge hydroxyl radicals.

  20. Enhancing the Photostability of Arylvinylenebipyridyl Compounds as Fluorescent Indicators for Intracellular Zinc(II) Ions

    PubMed Central

    Yuan, Zhao; Younes, Ali H.; Allen, John R.; Davidson, Michael W.; Zhu, Lei

    2015-01-01

    Arylvinylenebipyridyl (AVB) ligands are bright, zinc(II)-sensitive fluoroionophores. The applicability of AVBs as fluorescent indicators for imaging cellular zinc(II), however, is limited by low photostability, partially attributable to the photoisomerization of the vinylene functionality. Two configurationally immobilized (i.e., “locked”) AVB analogues are prepared in this work. The zinc(II)-sensitive photophysical properties and zinc(II) affinities of both AVBs and their locked analogues are characterized in organic and aqueous media. The zinc(II) sensitivity of the emission is attributed to the zinc(II)-dependent energies of the charge transfer excited states of these compounds. The configurationally locked ligands have improved photostability, while maintaining the brightness and zinc(II) sensibility of their AVB progenitors. The feasibility of the “locked” AVB analogues with improved photostability for imaging intracellular Zn(II) of eukaryotic cells using laser confocal fluorescence microscopy is demonstrated. PMID:25942357

  1. Fluorescence confocal endomicroscopy in biological imaging

    NASA Astrophysics Data System (ADS)

    Delaney, Peter; Thomas, Steven; Allen, John; McLaren, Wendy; Murr, Elise; Harris, Martin

    2007-02-01

    In vivo fluorescence microscopic imaging of biological systems in human disease states and animal models is possible with high optical resolution and mega pixel point-scanning performance using optimised off-the-shelf turn-key devices. There are however various trade-offs between tissue access and instrument performance when miniaturising in vivo microscopy systems. A miniature confocal scanning technology that was developed for clinical human endoscopy has been configured into a portable device for direct hand-held interrogation of living tissue in whole animal models (Optiscan FIVE-1 system). Scanning probes of 6.3mm diameter with a distal tip diameter of 5.0mm were constructed either in a 150mm length for accessible tissue, or a 300mm probe for laparoscopic interrogation of internal tissues in larger animal models. Both devices collect fluorescence confocal images (excitation 488 nm; emission >505 or >550 nm) comprised of 1024 x 1204 sampling points/image frame, with lateral resolution 0.7um; axial resolution 7um; FOV 475 x 475um. The operator can dynamically control imaging depth from the tissue surface to approx 250um in 4um steps via an internally integrated zaxis actuator. Further miniaturisation is achieved using an imaging contact probe based on scanning the proximal end of a high-density optical fibre bundle (~30,000 fibres) of <1mm diameter to transfer the confocal imaging plane to tissue in intact small animal organs, albeit at lower resolution (30,000 sampling points/image). In rodent models, imaging was performed using various fluorescent staining protocols including fluorescently labelled receptor ligands, labelled antibodies, FITC-dextrans, vital dyes and labelled cells administered topically or intravenously. Abdominal organs of large animals were accessed laparoscopically and contrasted using i.v. fluorescein-sodium. Articular cartilage of sheep and pigs was fluorescently stained with calcein-AM or fluorescein. Surface and sub-surface cellular and

  2. Theoretical investigation on ratiometric two-photon fluorescent probe for Zn2+ detection based on ICT mechanism

    NASA Astrophysics Data System (ADS)

    Huang, Shuang; Yang, Bao-Zhu; Ren, Ai-Min

    2016-06-01

    OPA (one-photon absorption), TPA (two-photon absorption) and fluorescence properties of a free ligand L upon coordination with Zn2+, and the regeneration with CN- were investigated in theory. According to our research, OPA spectra of ligand L show red-shift binding with Zn2+ while blue-shift with CN-. The fluorescence spectra and TPA wavelength are shifted in the same situation as those of OPA spectra. The value of TPA cross-section decreased at first, and then increased to 1813 GM for [L-Zn(CN)4]2-. Intramolecular charge transfer (ICT) mechanism was investigated by natural bond orbital (NBO) analysis. It demonstrates that L is hopeful to be a good ratiometric fluorescent probe for zinc ion detection in solution, and it can regenerate after CN- was introduced.

  3. Fluorescent metal ion chemosensors via cation exchange reactions of complexes, quantum dots, and metal-organic frameworks.

    PubMed

    Cheng, Jinghui; Zhou, Xiangge; Xiang, Haifeng

    2015-11-01

    Due to their wide range of applications and biological significance, fluorescent sensors have been an active research area in the past few years. In the present review, recent research developments on fluorescent chemosensors that detect metal ions via cation exchange reactions (transmetalation, metal displacement, or metal exchange reactions) of complexes, quantum dots, and metal-organic frameworks are described. These complex-based chemosensors might have a much better selectivity than the corresponding free ligands/receptors because of the shielding function of the filled-in metal ions. Moreover, not only the chemical structure of the ligands/receptors but also the identity of the central metal ions have a tremendous impact on the sensing performances. Therefore, sensing via cation exchange reactions potentially provides a new, simple, and powerful way to design fluorescent chemosensors. PMID:26375420

  4. Substrate-Dependent Ligand Inhibition of the Human Organic Cation Transporter OCT2

    PubMed Central

    Belzer, Mathew; Morales, Mark; Jagadish, Bhumasamudram; Mash, Eugene A.

    2013-01-01

    Organic cation transporter 2 (OCT2) mediates the initial step in renal secretion of organic cations: uptake from the blood, across the basolateral membrane, and into the renal proximal tubule cells. Because of its potential as a target for unwanted drug-drug interactions (DDIs), considerable attention has been directed toward understanding the basis of OCT2 selectivity. These studies typically assess selectivity based on ligand inhibition profiles for OCT2-mediated transport of a probe substrate. However, little attention has been given to the potential influence of the substrate on the profile of ligand inhibition. Here we compared the IC50 values obtained for a set of structurally distinct inhibitors against OCT2-mediated transport of three structurally distinct substrates: 1-methyl-4-phenylpyridinium (MPP); metformin; and a novel fluorescent substrate, N,N,N-trimethyl-2-[methyl(7-nitrobenzo[c][l,2,5]oxadiazol-4-yl)amino]ethanaminium iodide (NBD-MTMA). The median IC50 value for inhibition of MPP transport was 9-fold higher than that for inhibition of metformin transport. Similarly, the median IC50 value for inhibition of MPP transport was 5-fold higher than that for NBD-MTMA transport. However, this was not a systematic difference in inhibitory efficacy; the ratio of IC50 values, MPP versus NBD-MTMA, ranged from 88-fold (ipratropium) to 0.3-fold (metformin). These data show that 1) the choice of OCT2 substrate significantly influences both quantitative and qualitative inhibitory interactions with cationic drugs; and 2) ligand interactions with OCT2 are not restricted to competition for a common ligand binding site, consistent with a binding surface characterized by multiple, possibly overlapping interaction sites. Development of predictive models of DDIs with OCT2 must take into account the substrate dependence of ligand interaction with this protein. PMID:23709117

  5. The first scorpionate ligand based on diazaphosphole.

    PubMed

    Mlateček, Martin; Dostál, Libor; Růžičková, Zdeňka; Honzíček, Jan; Holubová, Jana; Erben, Milan

    2015-12-14

    The reaction of PhBCl2 with 1H-1,2,4-λ(3)-diazaphosphole in the presence of NEt3 gives a new scorpionate ligand, phenyl-tris(1,2,4-diazaphospholyl)borate (PhTdap). The coordination behaviour of this ligand toward transition and non-transition metals has been comprehensively studied. In the thallium(I) complex, Tl(PhTdap), κ(2)-N,N bonding supported with intramolecular η(3)-phenyl coordination has been observed in the solid state. Tl(PhTdap) also shows unusual intermolecular π-interactions between five-membered diazaphosphole rings and the thallium atom giving infinite molecular chains in the crystal. In the square planar complex [Pd(C,N-C6H4CH2NMe2)(PhTdap)], κ(2)-bonded scorpionate has been detected in both solution and in the solid state. For other studied compounds with the central metal ion Ti(IV), Mo(II), Mn(I), Fe(II), Ru(II), Co(II), Co(III), Ni(II) and Cd(II), the κ(3)-N,N,N coordination pattern was observed. Electronic properties of PhTdap and its ligand-field strength were elucidated from UV-Vis spectra of transition-metal species. The CH/P replacement on going from tris(pyrazolyl)borate to the ligand PhTdap causes a slight increase in electronic density rendered to the central metal atom. The following order of ligand-field strength has been established: HB(3,5-Me2pz)3 < PhB(pz)3 < HB(1,2,4-triazolyl) < HB(pz)3 < PhB(1,2,4-triazolyl) < PhTdap. The crystal structures of ten metal complexes bearing the new ligand are reported. The possibility of PhTdap coordination through the phosphorus atom is also briefly discussed. PMID:26537349

  6. Multifunctional Ligands in Transition Metal Catalysis (invited 'Focus' article),

    SciTech Connect

    Crabtree, Robert H

    2011-01-01

    Sophisticated ligands are now being designed that do far more than just fulfil their traditional spectator roles by binding to the metal and providing a sterically-defined binding pocket for the substrate in homogeneous transition metal catalysis. This Focus review emphasizes selected cases in which ligands carry additional functional groups that change the properties of the ligand as a result of an external stimulus or undergo catalytically-relevant ligand-based reactivity. These include proton responsive ligands capable of gaining or losing one or more protons, ligands having a hydrogen bonding function, electroresponsive ligands capable of gaining or losing one or more electrons, and photoresponsive ligands capable of undergoing a useful change of properties upon irradiation. Molecular recognition ligands and proton coupled electron transfer (PCET) are briefly discussed.

  7. Fluorescent Mu Selective Opioid Ligands from a Mixture Based Cyclic Peptide Library

    PubMed Central

    Li, Yangmei; Dooley, Colette T.; Misler, Jaime A.; Debevec, Ginamarie; Giulianotti, Marc A.; Cazares, Margaret E.; Maida, Laura; Houghten, Richard A.

    2013-01-01

    A positional scanning cyclic peptide library was generated using a penta-peptide thioester scaffold. Glycine was fixed at position R1. Diaminopropionic acid was fixed at position R3, with its γ-amino attaching to an anthraniloyl group. Positions R2 and R4 contained 36 L- and D- amino acids and position R5 contained 19 L- amino acids. Cyclization was performed in a mixture of acetonitrile and 1.5 M aqueous imidazole solution (7:1 v/v) at room temperature for 5 days. No significant cross-oligomerization was detected under the cyclization conditions. The library was screened in a binding assay for mu opioid receptor, identifying the active amino acid mixture at each position. A total of 40 individual cyclic peptides were identified and synthesized by the combinations of the most active amino acid mixtures found at three positions 5 × 4 × 2. Two cyclic peptides exhibited high binding affinities to opioid receptor. The most active cyclic peptide in the library was yielded to have Tyr at R2, D-Lys at R4 and Tyr at R5. Further investigation on this compound revealed the side chain-to-tail isomer to have greater binding affinity (14 nM) than the head-to-tail isomer (39 nM). Both isomers were selective for the mu-opioid receptor. PMID:23110623

  8. Monitoring cellular uptake and cytotoxicity of copper(II) complex using a fluorescent anthracene thiosemicarbazone ligand.

    PubMed

    Kate, Anup N; Kumbhar, Anupa A; Khan, Ayesha A; Joshi, Pranaya V; Puranik, Vedavati G

    2014-01-15

    The thiosemicarbazone derivative of anthracene (ATSC, anthracene thiosemicarbazone 1) and its copper(II) complex (CuATSC, 2) were synthesized and characterized by spectroscopic, electrochemical, and crystallographic techniques. Interaction of 1 and 2 with calf thymus (CT) DNA was explored using absorption and emission spectral methods, and viscosity measurements reveal a partial-intercalation binding mode. Their protein binding ability was monitored by the quenching of tryptophan emission using bovine serum albumin (BSA) as a model protein. Furthermore, their cellular uptake, in vitro cytotoxicity testing on the HeLa cell line, and flow cytometric analysis were carried out to ascertain the mode of cell death. Cell cycle analysis indicated that 1 and 2 cause cell cycle arrest in sub-G1 phase. PMID:24328322

  9. Development of the Novel PEG-PE-Based Polymer for the Reversible Attachment of Specific Ligands to Liposomes: Synthesis and in vitro Characterization

    PubMed Central

    Biswas, Swati; Dodwadkar, Namita S.; Sawant, Rupa R.; Torchilin, Vladimir P.

    2011-01-01

    Surface grafting of liposomes with the wide variety of ligands including antibodies and other proteins is a promising approach for targeted delivery of therapeutics. In this paper, we describe a simple method of synthesizing a hydrazine-functionalized polyethylene glycol-phosphatidylethanolamine (PEG-PE)-based amphiphilic polymer which can conjugate a variety of ligands via a reversible, pH-cleavable bond. In this method, the targeting ligand is attached to the distal end of the PEG chain, which facilitates its easy access to the targeted site of interaction. The reversible attachment of targeting ligands is useful especially in multifunctional liposomal systems, where after successfully performing the function of targeting to the specific site, the bulky ligands, such as proteins or antibodies, are cleaved off in response to an environmental stimulus to expose some other functionalities such as ligands for intracellular penetration or organelle-specific targeting. To investigate the applicability of the protocol, the model ligands monoclonal antinucleosome antibody 2C5 and antimyosin antibody 2G4, and glycoproteins concanavalin A (Con-A) and avidin were conjugated to the synthesized polymer and incorporated into liposomes. In vitro assays including biochemical, enzyme-linked immunosorbent, fluorescence microscopy and flow cytometry were used to confirm three key characteristics of the modified and/or liposome-attached proteins: successful conjugation of the targeting ligands to the polymer, preservation of specific activity of the ligands after the conjugation and liposome attachment, and the facile pH-sensitive ligand detachment. Monoclonal mAb 2C5 and 2G4, immobilized on the liposome surface, retained their binding affinity to corresponding antigens as confirmed by ELISA. The Con A-bearing liposomes showed significantly higher agglutination in the presence of its substrate mannan compared to plain liposomes (PL) and avidin-functionalized liposomes bound

  10. pH-responsive biocompatible fluorescent polymer nanoparticles based on phenylboronic acid for intracellular imaging and drug delivery

    NASA Astrophysics Data System (ADS)

    Li, Shengliang; Hu, Kelei; Cao, Weipeng; Sun, Yun; Sheng, Wang; Li, Feng; Wu, Yan; Liang, Xing-Jie

    2014-10-01

    To address current medical challenges, there is an urgent need to develop drug delivery systems with multiple functions, such as simultaneous stimuli-responsive drug release and real-time imaging. Biocompatible polymers have great potential for constructing smart multifunctional drug-delivery systems through grafting with other functional ligands. More importantly, novel biocompatible polymers with intrinsic fluorescence emission can work as theranostic nanomedicines for real-time imaging and drug delivery. Herein, we developed a highly fluorescent nanoparticle based on a phenylboronic acid-modified poly(lactic acid)-poly(ethyleneimine)(PLA-PEI) copolymer loaded with doxorubicin (Dox) for intracellular imaging and pH-responsive drug delivery. The nanoparticles exhibited superior fluorescence properties, such as fluorescence stability, no blinking and excitation-dependent fluorescence behavior. The Dox-loaded fluorescent nanoparticles showed pH-responsive drug release and were more effective in suppressing the proliferation of MCF-7 cells. In addition, the biocompatible fluorescent nanoparticles could be used as a tool for intracellular imaging and drug delivery, and the process of endosomal escape was traced by real-time imaging. These pH-responsive and biocompatible fluorescent polymer nanoparticles, based on phenylboronic acid, are promising tools for intracellular imaging and drug delivery.To address current medical challenges, there is an urgent need to develop drug delivery systems with multiple functions, such as simultaneous stimuli-responsive drug release and real-time imaging. Biocompatible polymers have great potential for constructing smart multifunctional drug-delivery systems through grafting with other functional ligands. More importantly, novel biocompatible polymers with intrinsic fluorescence emission can work as theranostic nanomedicines for real-time imaging and drug delivery. Herein, we developed a highly fluorescent nanoparticle based on a

  11. Quantitative approach of speleothems fluorescence

    NASA Astrophysics Data System (ADS)

    Quiers, Marine; Perrette, Yves; Poulenard, Jérôme; Chalmin, Emilie; Revol, Morgane

    2014-05-01

    In this study, we propose a framework to interpret quantitatively the fluorescence of speleothems organic matter (OM) by the way of a bank of water-extracted organic matter. Due to its efficiency to described dissolved organic matter (DOM) characteritics, fluorescence has been used to determined DOM signatures in natural systems, water circulations, OM transfer from soils, OM evolution in soils or recently, DOM changes in engineered treatment systems. Fluorescence has also been used in speleothems studies, mainly as a growth indicator. Only few studies interpret it as an environmental proxy. Indeed, the fluorescence of OM provides information on the type of organic molecules trapped in speleothems and their evolutions. But the most direct information given by fluorescence is the variation of OM quantities. Actually, increase of fluorescence intensity is generally related to an increase in OM quantity but may also be induced by calcite optical effect or qualitative change of OM. However, analytical technics used in water environments cannot be used for speleothem samples. In this study we propose to give a frame to interpret quantitatively the fluorescence signal of speleothems. 3 different samples of stalagmites from french northern Prealps were used. To allow the quantification of the fluorescence signal, we need to measure the fluorescence and the quantity of organic matter on the same sample. OM of speleothems was extracted by an acid digestion method and analysed with a spectrofluorimeter. However, it was not possible to quantify directly the OM, as the extract solvant was a high-concentrated acid. To solve this problem, a calibration using soil extracts was realised. Soils were chosen in order to represent the diversity of OM present in the environment above the caves. Attention was focused on soil and vegetation types, and landuse. Organic material was water extracted from soils and its fluorescence was also measured. Total organic carbon was performed on the

  12. Mechanism for attenuation of DNA binding by MarR family transcriptional regulators by small molecule ligands.

    PubMed

    Perera, Inoka C; Lee, Yong-Hwan; Wilkinson, Steven P; Grove, Anne

    2009-07-31

    Members of the multiple antibiotic resistance regulator (MarR) family control gene expression in a variety of metabolic processes in bacteria and archaea. Hypothetical uricase regulator (HucR), which belongs to the ligand-responsive branch of the MarR family, regulates uricase expression in Deinococcus radiodurans by binding a shared promoter region between uricase and HucR genes. We show here that HucR responds only to urate and, to a lesser extent, to xanthine by attenuated DNA binding, compared to other intermediates of purine degradation. Using molecular-dynamics-guided mutational analysis, we identified the ligand-binding site in HucR. Electrophoretic mobility shift assays and intrinsic Trp fluorescence have identified W20 from the N-terminal helix and R80 from helix 3, which serves as a scaffold for the DNA recognition helix, as being essential for ligand binding. Using structural data combined with in silico and in vitro analyses, we propose a mechanism for the attenuation of DNA binding in which a conformational change initiated by charge repulsion due to a bound ligand propagates to DNA recognition helices. This mechanism may apply generally to MarR homologs that bind anionic phenolic ligands. PMID:19501097

  13. A capillary electrophoresis method to explore the self-assembly of a novel polypeptide ligand with quantum dots.

    PubMed

    Wang, Jianhao; Zhang, Chencheng; Liu, Li; Kalesh, Karunakaran A; Qiu, Lin; Ding, Shumin; Fu, Minli; Gao, Li-Qian; Jiang, Pengju

    2016-08-01

    Polyhistidine peptides are effective ligands to coat quantum dots (QDs). It is known that both the number of histidine (His) residues repeats and their structural arrangements in a peptide ligand play important roles in the assembly of the peptide onto CdSe/ZnS QDs. However, due to steric hindrance, a peptide sequence with more than six His residue tandem repeats would hardly coordinate well with Zn(2+) in the QD shell to further enhance the binding affinity. To solve this problem, a His-containing peptide ligand, ATTO 590-E2 G (NH)6 (ATTO-NH), was specifically designed and synthesized for assembly with QDs. With sequential injection of QDs and ATTO-NH into the capillary electrophoresis with fluorescence detection, strong Förster resonance energy transfer phenomenon between the QDs and the ATTO 590 dye was observed, indicating efficient self-assembly of the novel peptide onto the QDs to form ATTO-NH capped QDs inside the capillary. The binding stability of the ligand onto the QD was then systematically investigated by titrating with imidazole, His, and a his-tag containing competitive peptide. It is believed that this new in-capillary assay significantly reduced the sample consumption and the analysis time. By functionalizing QDs with certain metal cation-specific group fused peptide ligand, the QD-based probes could be even extended to the online detection of metal cations for monitoring environment in the future. PMID:27334251

  14. Characterizing the Effect of Multivalent Conjugates Composed of Aβ-Specific Ligands and Metal Nanoparticles on Neurotoxic Fibrillar Aggregation.

    PubMed

    Streich, Carmen; Akkari, Laura; Decker, Christina; Bormann, Jenny; Rehbock, Christoph; Müller-Schiffmann, Andreas; Niemeyer, Felix Carlsson; Nagel-Steger, Luitgard; Willbold, Dieter; Sacca, Barbara; Korth, Carsten; Schrader, Thomas; Barcikowski, Stephan

    2016-08-23

    Therapeutically active small molecules represent promising nonimmunogenic alternatives to antibodies for specifically targeting disease-relevant receptors. However, a potential drawback compared to antibody-antigen interactions may be the lower affinity of small molecules toward receptors. Here, we overcome this low-affinity problem by coating the surface of nanoparticles (NPs) with multiple ligands. Specifically, we explored the use of gold and platinum nanoparticles to increase the binding affinity of Aβ-specific small molecules to inhibit Aβ peptide aggregation into fibrils in vitro. The interactions of bare NPs, free ligands, and NP-bound ligands with Aβ are comprehensively studied via physicochemical methods (spectroscopy, microscopy, immunologic tests) and cell assays. Reduction of thioflavin T fluorescence, as an indicator for β-sheet content, and inhibition of cellular Aβ excretion are even more effective with NP-bound ligands than with the free ligands. The results from this study may have implications in the development of therapeutics for treating Alzheimer's disease. PMID:27404114

  15. Strong Ligand-Protein Interactions Derived from Diffuse Ligand Interactions with Loose Binding Sites.

    PubMed

    Marsh, Lorraine

    2015-01-01

    Many systems in biology rely on binding of ligands to target proteins in a single high-affinity conformation with a favorable ΔG. Alternatively, interactions of ligands with protein regions that allow diffuse binding, distributed over multiple sites and conformations, can exhibit favorable ΔG because of their higher entropy. Diffuse binding may be biologically important for multidrug transporters and carrier proteins. A fine-grained computational method for numerical integration of total binding ΔG arising from diffuse regional interaction of a ligand in multiple conformations using a Markov Chain Monte Carlo (MCMC) approach is presented. This method yields a metric that quantifies the influence on overall ligand affinity of ligand binding to multiple, distinct sites within a protein binding region. This metric is essentially a measure of dispersion in equilibrium ligand binding and depends on both the number of potential sites of interaction and the distribution of their individual predicted affinities. Analysis of test cases indicates that, for some ligand/protein pairs involving transporters and carrier proteins, diffuse binding contributes greatly to total affinity, whereas in other cases the influence is modest. This approach may be useful for studying situations where "nonspecific" interactions contribute to biological function. PMID:26064949

  16. Strong Ligand-Protein Interactions Derived from Diffuse Ligand Interactions with Loose Binding Sites

    PubMed Central

    2015-01-01

    Many systems in biology rely on binding of ligands to target proteins in a single high-affinity conformation with a favorable ΔG. Alternatively, interactions of ligands with protein regions that allow diffuse binding, distributed over multiple sites and conformations, can exhibit favorable ΔG because of their higher entropy. Diffuse binding may be biologically important for multidrug transporters and carrier proteins. A fine-grained computational method for numerical integration of total binding ΔG arising from diffuse regional interaction of a ligand in multiple conformations using a Markov Chain Monte Carlo (MCMC) approach is presented. This method yields a metric that quantifies the influence on overall ligand affinity of ligand binding to multiple, distinct sites within a protein binding region. This metric is essentially a measure of dispersion in equilibrium ligand binding and depends on both the number of potential sites of interaction and the distribution of their individual predicted affinities. Analysis of test cases indicates that, for some ligand/protein pairs involving transporters and carrier proteins, diffuse binding contributes greatly to total affinity, whereas in other cases the influence is modest. This approach may be useful for studying situations where “nonspecific” interactions contribute to biological function. PMID:26064949

  17. Permethrin and its metabolites affect Cu/Zn superoxide conformation: fluorescence and in silico evidences.

    PubMed

    Rosita, Gabbianelli; Manuel, Carloni; Franco, Marmocchi; Cinzia, Nasuti; Donatella, Fedeli; Emiliano, Laudadio; Luca, Massaccesi; Roberta, Galeazzi

    2015-01-01

    The proclivity of permethrin and its metabolites to affect the structure and activity of Cu/Zn superoxide dismutase (SOD) has been investigated by using intrinsic fluorescence and 8-ANS fluorescence techniques. In silico molecular docking investigations were carried out in order to assess the means of interaction at a molecular level between SOD and the considered ligands. Results show that both, permethrin and its metabolites are able to induce conformational variation on SOD. Permethrin and 3-phenoxybenzyl alcohol metabolite induce a blue shift toward the hydrophobic amino acids Leu-101, Ile-102, Leu-104, Ile-110 and Ile-111, with a significant peak increase. An opposite effect was shown by 3-phenoxy benzaldehyde and 3-phenoxybenzoic acid with a progressive reduction of tyrosine fluorescence emission, without any shift. Computational findings confirm that all the molecules considered have more than one allosteric binding site but none of them interact with SOD at its catalytic Cu/Zn cleft. Moreover, all the binding poses found are very close in binding energy thus demonstrating that there is not only a preferred interaction site but most of them are important due to their relative energy in equilibrium with a population strictly connected to the ligand concentration. In the obtained complexes, all the ligands are involved in many hydrogen bonds through their polar oxygen moieties but due to the presence of a common aromatic hydrophobic core, many hydrophobic interactions are due to the SOD nature rich in apolar amino acids. Furthermore, for each ligand it can be pointed out the presence of a highly populated docked structure with a specific interaction of permethrin and its metabolites with Tyr-108, responsible for changes in fluorescence emission. PMID:25354707

  18. Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo

    PubMed Central

    Plamont, Marie-Aude; Billon-Denis, Emmanuelle; Maurin, Sylvie; Gauron, Carole; Pimenta, Frederico M.; Specht, Christian G.; Shi, Jian; Quérard, Jérôme; Pan, Buyan; Rossignol, Julien; Moncoq, Karine; Morellet, Nelly; Volovitch, Michel; Lescop, Ewen; Chen, Yong; Triller, Antoine; Vriz, Sophie; Le Saux, Thomas; Jullien, Ludovic; Gautier, Arnaud

    2016-01-01

    This paper presents Yellow Fluorescence-Activating and absorption-Shifting Tag (Y-FAST), a small monomeric protein tag, half as large as the green fluorescent protein, enabling fluorescent labeling of proteins in a reversible and specific manner through the reversible binding and activation of a cell-permeant and nontoxic fluorogenic ligand (a so-called fluorogen). A unique fluorogen activation mechanism based on two spectroscopic changes, increase of fluorescence quantum yield and absorption red shift, provides high labeling selectivity. Y-FAST was engineered from the 14-kDa photoactive yellow protein by directed evolution using yeast display and fluorescence-activated cell sorting. Y-FAST is as bright as common fluorescent proteins, exhibits good photostability, and allows the efficient labeling of proteins in various organelles and hosts. Upon fluorogen binding, fluorescence appears instantaneously, allowing monitoring of rapid processes in near real time. Y-FAST distinguishes itself from other tagging systems because the fluorogen binding is highly dynamic and fully reversible, which enables rapid labeling and unlabeling of proteins by addition and withdrawal of the fluorogen, opening new exciting prospects for the development of multiplexing imaging protocols based on sequential labeling. PMID:26711992

  19. Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo.

    PubMed

    Plamont, Marie-Aude; Billon-Denis, Emmanuelle; Maurin, Sylvie; Gauron, Carole; Pimenta, Frederico M; Specht, Christian G; Shi, Jian; Quérard, Jérôme; Pan, Buyan; Rossignol, Julien; Moncoq, Karine; Morellet, Nelly; Volovitch, Michel; Lescop, Ewen; Chen, Yong; Triller, Antoine; Vriz, Sophie; Le Saux, Thomas; Jullien, Ludovic; Gautier, Arnaud

    2016-01-19

    This paper presents Yellow Fluorescence-Activating and absorption-Shifting Tag (Y-FAST), a small monomeric protein tag, half as large as the green fluorescent protein, enabling fluorescent labeling of proteins in a reversible and specific manner through the reversible binding and activation of a cell-permeant and nontoxic fluorogenic ligand (a so-called fluorogen). A unique fluorogen activation mechanism based on two spectroscopic changes, increase of fluorescence quantum yield and absorption red shift, provides high labeling selectivity. Y-FAST was engineered from the 14-kDa photoactive yellow protein by directed evolution using yeast display and fluorescence-activated cell sorting. Y-FAST is as bright as common fluorescent proteins, exhibits good photostability, and allows the efficient labeling of proteins in various organelles and hosts. Upon fluorogen binding, fluorescence appears instantaneously, allowing monitoring of rapid processes in near real time. Y-FAST distinguishes itself from other tagging systems because the fluorogen binding is highly dynamic and fully reversible, which enables rapid labeling and unlabeling of proteins by addition and withdrawal of the fluorogen, opening new exciting prospects for the development of multiplexing imaging protocols based on sequential labeling. PMID:26711992

  20. Fluorescence of quantum dots on e-beam patterned and DNA origami substrates

    NASA Astrophysics Data System (ADS)

    Corrigan, Timothy D.; Kessinger, Matthew; Kidd, Jesse; Neff, David; Rahman, Masudur; Norton, Michael L.

    2015-05-01

    Attachment of quantum dots or fluorescent molecules to gold nanoparticles has a variety of optical labeling and sensory applications. In this study, we use both e-beam lithography and DNA origami to examine the fluorescence enhancement of fluorescent molecules and quantum dots with a systematic approach to understanding the contribution of gold nanoparticle size and interparticle spacing. The unique design of our patterns allows us to study the effects of size and spacing of the gold nanoparticles on the enhancement of fluorescence in one quick study with constant conditions - removing undesirable effects such as differences in concentration of quantum dots or other chemistry differences that plague multiple experiments. We also discuss the fluorescence and bonding of CdSe/ZnS quantum dots to both gold as well as DNA for use in self assembled DNA constructs. Specifically, bioconjugated CdSe/ZnS core/shell quantum dots were synthesized and functionalized with MPA using both traditional ligand exchange as well as newly developed in situ functionalization techniques used to increase the quantum yield of the quantum dots. We will present fluorescent images showing results of optimal size and spacing for fluorescence as well as demonstrating attachment chemistry of the quantum dots.

  1. Microwave-assisted synthesis of water-soluble, fluorescent gold nanoclusters capped with small organic molecules and a revealing fluorescence and X-ray absorption study

    NASA Astrophysics Data System (ADS)

    Helmbrecht, C.; Lützenkirchen-Hecht, D.; Frank, W.

    2015-03-01

    Colourless solutions of blue light-emitting, water-soluble gold nanoclusters (AuNC) were synthesized from gold colloids under microwave irradiation using small organic molecules as ligands. Stabilized by 1,3,5-triaza-7-phosphaadamantane (TPA) or l-glutamine (GLU), fluorescence quantum yields up to 5% were obtained. AuNC are considered to be very promising for biological labelling, optoelectronic devices and light-emitting materials but the structure-property relationships have still not been fully clarified. To expand the knowledge about the AuNC apart from their fluorescent properties they were studied by X-ray absorption spectroscopy elucidating the oxidation state of the nanoclusters' gold atoms. Based on curve fitting of the XANES spectra in comparison to several gold references, optically transparent fluorescent AuNC are predicted to be ligand-stabilized Au5+ species. Additionally, their near edge structure compared with analogous results of polynuclear clusters known from the literature discloses an increasing intensity of the feature close to the absorption edge with decreasing cluster size. As a result, a linear relationship between the cluster size and the X-ray absorption coefficient can be established for the first time.Colourless solutions of blue light-emitting, water-soluble gold nanoclusters (AuNC) were synthesized from gold colloids under microwave irradiation using small organic molecules as ligands. Stabilized by 1,3,5-triaza-7-phosphaadamantane (TPA) or l-glutamine (GLU), fluorescence quantum yields up to 5% were obtained. AuNC are considered to be very promising for biological labelling, optoelectronic devices and light-emitting materials but the structure-property relationships have still not been fully clarified. To expand the knowledge about the AuNC apart from their fluorescent properties they were studied by X-ray absorption spectroscopy elucidating the oxidation state of the nanoclusters' gold atoms. Based on curve fitting of the XANES

  2. Synthesis, characterization, DNA/protein interaction and cytotoxicity studies of Cu(II) and Co(II) complexes derived from dipyridyl triazole ligands

    NASA Astrophysics Data System (ADS)

    Zhang, Wei; Yao, Di; Wei, Yi; Tang, Jie; Bian, He-Dong; Huang, Fu-Ping; Liang, Hong

    2016-06-01

    Four different transition metal complexes containing dipyridyl triazole ligands, namely [Cu(abpt)2Cl2]·2H2O (1), [Cu(abpt)2(ClO4)2] (2), [Co2(abpt)2(H2O)2Cl2]·Cl2·4H2O (3) and [Co2(Hbpt)2(CH3OH)2(NO3)2] (4) have been designed, synthesized and further structurally characterized by X-ray crystallography, ESI-MS, elemental analysis, IR and Raman spectroscopy. In these complexes, the both ligands act as bidentate ligands with N, N donors. DNA binding interactions with calf thymus DNA (ct-DNA) of the ligand and its complexes 1 ~ 4 were investigated via electronic absorption, fluorescence quenching, circular dichroism and viscosity measurements as well as confocal Laser Raman spectroscopy. The results show these complexes are able to bind to DNA via the non-covalent mode i.e. intercalation and groove binding or electrostatic interactions. The interactions with bovine serum albumin (BSA) were also studied using UV-Vis and fluorescence spectroscopic methods which indicated that fluorescence quenching of BSA by these compounds was the presence of both static and dynamic quenching. Moreover, the in vitro cytotoxic effects of the complexes against four cell lines SK-OV-3, HL-7702, BEL7404 and NCI-H460 showed the necessity of the coordination action on the biological properties on the respective complex and that all four complexes exhibited substantial cytotoxic activity.

  3. Mononuclear, dinuclear and 1-D polymeric complexes of Cd(II) of a pyridyl pyrazole ligand: Syntheses, crystal structures and photoluminescence studies

    NASA Astrophysics Data System (ADS)

    Das, Kinsuk; Konar, Saugata; Jana, Atanu; Barik, Anil Kumar; Roy, Sangita; Kar, Susanta Kumar

    2013-03-01

    The syntheses, crystal structures and photoluminescence properties of four new Cd(II) complexes are reported using strongly coordinating ligand 3,5-dimethyl-1-(2'-pyridyl) pyrazole (L) in presence of anionic ancillary bridging ligands as nitrite, chloride and dicyanamide. Among the complexes two (1 and 2) are monomeric, 3 is μ2 - chloro bridged dimer and the last one (4) is a mixed alternate chloro - end to end (EE) dicyanamide bridged 1D polymer. All the four complexes have been X-ray crystallographically characterized. The ligand L behaves as a potent bidentate neutral N, N donor. Geometrical diversity of Cd(II) complexes is due to no loss or gain of crystal field stability with the variation of geometry. Consequently the stability of a structure depends on steric requirements. The ligand L shows considerable fluorescence and all four complexes in methanol exhibit interesting photoluminescence properties with different emission intensities. The band maxima and fluorescence efficiency (in methanol) are found to be dependent on the coordination chromophore and structural rigidity induced by the incorporated Cd(II) ion. Among the synthesized complexes 1 exhibits the highest fluorescence intensity in methanol.

  4. Fluorescence Reveals Contamination From Adhesives

    NASA Technical Reports Server (NTRS)

    Nikolia, William

    1992-01-01

    Contamination of nearby surfaces from ingredients in some adhesive materials detected by ultraviolet illumination and observation of resulting fluorescence. Identification of contaminants via telltale fluorescence not new; rather, significance lies in method of implementation and potential extension to wider variety of materials and applications.

  5. Assessing Photosynthesis by Fluorescence Imaging

    ERIC Educational Resources Information Center

    Saura, Pedro; Quiles, Maria Jose

    2011-01-01

    This practical paper describes a novel fluorescence imaging experiment to study the three processes of photochemistry, fluorescence and thermal energy dissipation, which compete during the dissipation of excitation energy in photosynthesis. The technique represents a non-invasive tool for revealing and understanding the spatial heterogeneity in…

  6. Frequency domain fluorescence lifetime microwell-plate platform for respirometry measurements

    NASA Astrophysics Data System (ADS)

    Chatni, M. R.; Yale, G.; Van Ryckeghem, A.; Porterfield, D. M.

    2010-04-01

    Traditionally micro-well plate based platforms used in biology utilize fluorescence intensity based methods to measure processes of biological relevance. However, fluorescence intensity measurements suffer from calibration drift due to a variety of factors. Photobleaching and self-quenching of the fluorescent dyes cause the intensity signal to drop over the lifetime of sensor immobilized inside the well. Variation in turbidity of the sample during the course of the measurement affects the measured fluorescence intensity. In comparison, fluorescence lifetime measurements are not significantly affected by these factors because fluorescence lifetime is a physico-chemical property of the fluorescent dye. Reliable and inexpensive frequency domain fluorescence lifetime instrumentation platforms are possible because the greater tolerance for optical alignment, and because they can be performed using inexpensive light sources such as LEDs. In this paper we report the development of a frequency domain fluorescence lifetime well-plate platform utilizing an oxygen sensitive transition-metal ligand complex fluorophore with a lifetime in the microsecond range. The fluorescence lifetime dye is incorporated in a polymer matrix and immobilized on the base of micro-well of a 60 well micro-well plate. Respiration measurements are performed in both aqueous and non-aqueous environment. Respirometry measurements were recorded from single Daphnia magna egg in hard water. Daphnia is an aquatic organism, important in environmental toxicology as a standard bioassay and early warning indicator for water quality monitoring. Also respirometry measurements were recorded from Tribolium castaneum eggs, which are common pests in the processed flour industry. These eggs were subjected to mitochondrial electron transport chain inhibitor such as potassium cyanide (KCN) and its effects on egg respiration were measured in real-time.

  7. A sole multi-analyte receptor responds with three distinct fluorescence signals: traffic signal like sensing of Al(3+), Zn(2+) and F(-).

    PubMed

    Datta, Barun Kumar; Thiyagarajan, Durairaj; Ramesh, Aiyagari; Das, Gopal

    2015-08-01

    A dialdehyde-based multi-analyte sensor renders distinctive emission spectra for Al(3+), Zn(2+) and F(-) ions. The ligand exhibited different types of interactions with these three different ions resulting in the enhancement of fluorescence intensity at three different wavelengths. All the sensing processes were studied in detail by absorption spectroscopy, emission spectroscopy and (1)H-NMR titration experiment. The ligand has the working ability in a wide pH range including the physiological pH. The ligand is non-toxic and amicable for sensing intracellular Al(3+) and Zn(2+) in live HeLa cells. PMID:26107138

  8. Fiber-optic fluorescence imaging

    PubMed Central

    Flusberg, Benjamin A; Cocker, Eric D; Piyawattanametha, Wibool; Jung, Juergen C; Cheung, Eunice L M; Schnitzer, Mark J

    2010-01-01

    Optical fibers guide light between separate locations and enable new types of fluorescence imaging. Fiber-optic fluorescence imaging systems include portable handheld microscopes, flexible endoscopes well suited for imaging within hollow tissue cavities and microendoscopes that allow minimally invasive high-resolution imaging deep within tissue. A challenge in the creation of such devices is the design and integration of miniaturized optical and mechanical components. Until recently, fiber-based fluorescence imaging was mainly limited to epifluorescence and scanning confocal modalities. Two new classes of photonic crystal fiber facilitate ultrashort pulse delivery for fiber-optic two-photon fluorescence imaging. An upcoming generation of fluorescence imaging devices will be based on microfabricated device components. PMID:16299479

  9. Fluorescence Polarization Assays in Small Molecule Screening

    PubMed Central

    Lea, Wendy A.; Simeonov, Anton

    2011-01-01

    Importance of the field Fluorescence polarization (FP) is a homogeneous method that allows rapid and quantitative analysis of diverse molecular interactions and enzyme activities. This technique has been widely utilized in clinical and biomedical settings, including the diagnosis of certain diseases and monitoring therapeutic drug levels in body fluids. Recent developments in the field has been symbolized by the facile adoption of FP in high-throughput screening (HTS) and small molecule drug discovery of an increasing range of target classes. Areas covered in this review The article provides a brief overview on the theoretical foundation of FP, followed by updates on recent advancements in its application for various drug target classes, including G-protein coupled receptors (GPCRs), enzymes and protein-protein interactions (PPIs). The strengths and weaknesses of this method, practical considerations in assay design, novel applications, and future directions are also discussed. What the reader will gain The reader will be informed of the most recent advancements and future directions of FP application to small molecule screening. Take home message In addition to its continued utilization in high-throughput screening, FP has expanded into new disease and target areas and has been marked by increased use of labeled small molecule ligands for receptor binding studies. PMID:22328899

  10. Determination of Ligand Pathways in Globins

    PubMed Central

    Salter, Mallory D.; Blouin, George C.; Soman, Jayashree; Singleton, Eileen W.; Dewilde, Sylvia; Moens, Luc; Pesce, Alessandra; Nardini, Marco; Bolognesi, Martino; Olson, John S.

    2012-01-01

    Although molecular dynamics simulations suggest multiple interior pathways for O2 entry into and exit from globins, most experiments indicate well defined single pathways. In 2001, we highlighted the effects of large-to-small amino acid replacements on rates for ligand entry and exit onto the three-dimensional structure of sperm whale myoglobin. The resultant map argued strongly for ligand movement through a short channel from the heme iron to solvent that is gated by the distal histidine (His-64(E7)) near the solvent edge of the porphyrin ring. In this work, we have applied the same mutagenesis mapping strategy to the neuronal mini-hemoglobin from Cerebratulus lacteus (CerHb), which has a large internal tunnel from the heme iron to the C-terminal ends of the E and H helices, a direction that is 180° opposite to the E7 channel. Detailed comparisons of the new CerHb map with expanded results for Mb show unambiguously that the dominant (>90%) ligand pathway in CerHb is through the internal tunnel, and the major (>75%) ligand pathway in Mb is through the E7 gate. These results demonstrate that: 1) mutagenesis mapping can identify internal pathways when they exist; 2) molecular dynamics simulations need to be refined to address discrepancies with experimental observations; and 3) alternative pathways have evolved in globins to meet specific physiological demands. PMID:22859299

  11. MOESSBAUER EFFECT IN HEMOGLOBIN WITH DIFFERENT LIGANDS.

    PubMed

    GONSER, U; GRANT, R W; KREGZDE, J

    1964-02-14

    Recoil-free nuclear gamma-ray resonance adsorption was observed in the iron-57 of blood. The spectral parameters are dependent on the ligand bound to the iron atoms in hemoglobin. The results are interpreted in terms of isomeric shifts and quad rupole splittings. PMID:14081237

  12. Identification of ligands for bacterial sensor proteins.

    PubMed

    Fernández, Matilde; Morel, Bertrand; Corral-Lugo, Andrés; Rico-Jiménez, Miriam; Martín-Mora, David; López-Farfán, Diana; Reyes-Darias, José Antonio; Matilla, Miguel A; Ortega, Álvaro; Krell, Tino

    2016-02-01

    Bacteria have evolved a variety of different signal transduction mechanisms. However, the cognate signal molecule for the very large amount of corresponding sensor proteins is unknown and their functional annotation represents a major bottleneck in the field of signal transduction. The knowledge of the signal molecule is an essential prerequisite to understand the signalling mechanisms. Recently, the identification of signal molecules by the high-throughput protein screening of commercially available ligand collections using differential scanning fluorimetry has shown promise to resolve this bottleneck. Based on the analysis of a significant number of different ligand binding domains (LBDs) in our laboratory, we identified two issues that need to be taken into account in the experimental design. Since a number of LBDs require the dimeric state for ligand recognition, it has to be assured that the protein analysed is indeed in the dimeric form. A number of other examples demonstrate that purified LBDs can contain bound ligand which prevents further binding. In such cases, the apo-form can be generated by denaturation and subsequent refolding. We are convinced that this approach will accelerate the functional annotation of sensor proteins which will help to understand regulatory circuits in bacteria. PMID:26511375

  13. Micropatterned Surfaces with Controlled Ligand Tethering

    PubMed Central

    Petrie, Timothy A.; Stanley, Brandon T.; García, Andrés J.

    2008-01-01

    Microcontact printing (μ-CP) is a facile, cost-effective, and versatile soft-lithography technique to create 2-dimensional patterns of domains with distinct functionalities that provides a robust platform to generate micropatterned biotechnological arrays and cell culture substrates. Current μ-CP approaches rely on non-specific immobilization of biological ligands, either by direct printing or adsorption from solution, onto micropatterned domains surrounded by a non-fouling background. This technique is limited by insufficient control over ligand density. We present a modified μ-CP protocol involving stamping mixed ratios of carboxyl- and tri(ethylene glycol)-terminated alkanethiols that provides for precise covalent tethering of single or multiple ligands to prescribed micropatterns via standard peptide chemistry. Processing parameters were optimized to identify conditions that control relevant endpoint pattern characteristics. This technique provides a facile method to generate micropatterned arrays with tailorable and controlled presentation of biological ligands for biotechnological applications and analyses of cell-material interactions. PMID:18570314

  14. Nanoparticle ligand presentation for targeting solid tumors.

    PubMed

    Duskey, Jason T; Rice, Kevin G

    2014-10-01

    Among the many scientific advances to come from the study of nanoscience, the development of ligand-targeted nanoparticles to eliminate solid tumors is predicted to have a major impact on human health. There are many reports describing novel designs and testing of targeted nanoparticles to treat cancer. While the principles of the technology are well demonstrated in controlled lab experiments, there are still many hurdles to overcome for the science to mature into truly efficacious targeted nanoparticles that join the arsenal of agents currently used to treat cancer in humans. One of these hurdles is overcoming unwanted biodistribution to the liver while maximizing delivery to the tumor. This almost certainly requires advances in both nanoparticle stealth technology and targeting. Currently, it continues to be a challenge to control the loading of ligands onto polyethylene glycol (PEG) to achieve maximal targeting. Nanoparticle cellular uptake and subcellular targeting of genes and siRNA also remain a challenge. This review examines the types of ligands that have been most often used to target nanoparticles to solid tumors. As the science matures over the coming decade, careful control over ligand presentation on nanoparticles of precise size, shape, and charge will likely play a major role in achieving success. PMID:24927668

  15. Ligand configurational entropy and protein binding.

    PubMed

    Chang, Chia-en A; Chen, Wei; Gilson, Michael K

    2007-01-30

    The restriction of a small molecule's motion on binding to a protein causes a loss of configurational entropy, and thus a penalty in binding affinity. Some energy models used in computer-aided ligand design neglect this entropic penalty, whereas others account for it based on an expected drop in the number of accessible rotamers upon binding. However, the validity of the physical assumptions underlying the various approaches is largely unexamined. The present study addresses this issue by using Mining Minima calculations to analyze the association of amprenavir with HIV protease. The computed loss in ligand configurational entropy is large, contributing approximately 25 kcal/mol (4.184 kJ/kcal) to DeltaG degrees. Most of this loss results from narrower energy wells in the bound state, rather than a drop in the number of accessible rotamers. Coupling among rotation/translation and internal degrees of freedom complicates the decomposition of the entropy change into additive terms. The results highlight the potential to gain affinity by designing conformationally restricted ligands and have implications for the formulation of energy models for ligand scoring. PMID:17242351

  16. Ligand iron catalysts for selective hydrogenation

    DOEpatents

    Casey, Charles P.; Guan, Hairong

    2010-11-16

    Disclosed are iron ligand catalysts for selective hydrogenation of aldehydes, ketones and imines. A catalyst such as dicarbonyl iron hydride hydroxycyclopentadiene) complex uses the OH on the five member ring and hydrogen linked to the iron to facilitate hydrogenation reactions, particularly in the presence of hydrogen gas.

  17. Ligand configurational entropy and protein binding

    PubMed Central

    Chang, Chia-en A.; Chen, Wei; Gilson, Michael K.

    2007-01-01

    The restriction of a small molecule's motion on binding to a protein causes a loss of configurational entropy, and thus a penalty in binding affinity. Some energy models used in computer-aided ligand design neglect this entropic penalty, whereas others account for it based on an expected drop in the number of accessible rotamers upon binding. However, the validity of the physical assumptions underlying the various approaches is largely unexamined. The present study addresses this issue by using Mining Minima calculations to analyze the association of amprenavir with HIV protease. The computed loss in ligand configurational entropy is large, contributing ∼25 kcal/mol (4.184 kJ/kcal) to ΔG°. Most of this loss results from narrower energy wells in the bound state, rather than a drop in the number of accessible rotamers. Coupling among rotation/translation and internal degrees of freedom complicates the decomposition of the entropy change into additive terms. The results highlight the potential to gain affinity by designing conformationally restricted ligands and have implications for the formulation of energy models for ligand scoring. PMID:17242351

  18. Evaluation of DNA binding, DNA cleavage, protein binding, radical scavenging and in vitro cytotoxic activities of ruthenium(II) complexes containing 2,4-dihydroxy benzylidene ligands.

    PubMed

    Mohanraj, Maruthachalam; Ayyannan, Ganesan; Raja, Gunasekaran; Jayabalakrishnan, Chinnasamy

    2016-12-01

    The new ruthenium(II) complexes with hydrazone ligands, 4-Methyl-benzoic acid (2,4-dihydroxy-benzylidene)-hydrazide (HL(1)), 4-Methoxy-benzoic acid (2,4-dihydroxy-benzylidene)-hydrazide (HL(2)), 4-Bromo-benzoic acid (2,4-dihydroxy-benzylidene)-hydrazide (HL(3)), were synthesized and characterized by various spectro analytical techniques. The molecular structures of the ligands were confirmed by single crystal X-ray diffraction technique. The DNA binding studies of the ligands and complexes were examined by absorption, fluorescence, viscosity and cyclic voltammetry methods. The results indicated that the ligands and complexes could interact with calf thymus DNA (CT-DNA) through intercalation. The DNA cleavage activity of the complexes was evaluated by gel electrophoresis assay, which revealed that the complexes are good DNA cleaving agents. The binding interaction of the ligands and complexes with bovine serum albumin (BSA) was investigated using fluorescence spectroscopic method. Antioxidant studies showed that the complexes have a strong radical scavenging properties. Further, the cytotoxic effect of the complexes examined on cancerous cell lines showed that the complexes exhibit significant anticancer activity. PMID:27612830

  19. Exogenous specific fluorescence marker location reconstruction using surface fluorescence imaging

    NASA Astrophysics Data System (ADS)

    Avital, Garashi; Gannot, Israel; Chernomordik, Victor V.; Gannot, Gallya; Gandjbakhche, Amir H.

    2003-07-01

    Diseased tissue may be specifically marked by an exogenous fluorescent marker and then, following laser activation of the marker, optically and non-invasively detected through fluorescence imaging. Interaction of a fluorophore, conjugated to an appropriate antibody, with the antigen expressed by the diseased tissue, can indicate the presence of a specific disease. Using an optical detection system and a reconstruction algorithm, we were able to determine the fluorophore"s position in the tissue. We present 3D reconstructions of the location of a fluorescent marker, FITC, in the tongues of mice. One group of BALB/c mice was injected with squamous cell carcinoma (SqCC) cell line to the tongue, while another group served as the control. After tumor development, the mice"s tongues were injected with FITC conjugated to anti-CD3 and anti-CD 19 antibodies. An Argon laser excited the marker at 488 nm while a high precision fluorescent camera collected the emitted fluorescence. Measurements were performed with the fluorescent marker embedded at various simulated depths. The simulation was performed using agarose-based gel slabs applied to the tongue as tissue-like phantoms. A biopsy was taken from every mouse after the procedure and the excised tissue was histologically evaluated. We reconstruct the fluorescent marker"s location in 3D using an algorithm based on the random walk theory.

  20. Photouncaging nanoparticles for MRI and fluorescence imaging in vitro and in vivo.

    PubMed

    Shibu, Edakkattuparambil S; Ono, Kenji; Sugino, Sakiko; Nishioka, Ayami; Yasuda, Akikazu; Shigeri, Yasushi; Wakida, Shin-Ichi; Sawada, Makoto; Biju, Vasudevanpillai

    2013-11-26

    Multimodal and multifunctional nanomaterials are promising candidates for bioimaging and therapeutic applications in the nanomedicine settings. Here we report the preparation of photouncaging nanoparticles with fluorescence and magnetic modalities and evaluation of their potentials for in vitro and in vivo bioimaging. Photoactivation of such bimodal nanoparticles prepared using photouncaging ligands, CdSe/ZnS quantum dots, and super paramagnetic iron oxide nanoparticles results in the systematic uncaging of the particles, which is correlated with continuous changes in the absorption, mass and NMR spectra of the ligands. Fluorescence and magnetic components of the bimodal nanoparticles are characterized using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and elemental analyses using energy dispersive X-ray (EDX) spectroscopy and X-ray photoelectron spectroscopy (XPS). Bioconjugation of the nanoparticles with peptide hormones renders them with biocompatibility and efficient intracellular transport as seen in the fluorescence and MRI images of mouse melanoma cells (B16) or human lung epithelial adenocarcinoma cells (H1650). Biocompatibility of the nanoparticles is evaluated using MTT cytotoxicity assays, which show cell viability over 90%. Further, we combine MRI and NIR fluorescence imaging in C57BL/6 (B6) mice subcutaneously or intravenously injected with the photouncaging nanoparticles and follow the in vivo fate of the nanoparticles. Interestingly, the intravenously injected nanoparticles initially accumulate in the liver within 30 min post injection and subsequently clear by the renal excretion within 48 h as seen in the time-dependent MRI and fluorescence images of the liver, urinary bladder, and urine samples. Photouncaging ligands such as the ones reported in this article are promising candidates for not only the site-specific delivery of nanomaterials-based contrast agents and drugs but also the systematic uncaging and renal

  1. Prospect of Bioflavonoid Fisetin as a Quadruplex DNA Ligand: A Biophysical Approach

    PubMed Central

    Sengupta, Bidisha; Pahari, Biswapathik; Blackmon, Laura; Sengupta, Pradeep K.

    2013-01-01

    Quadruplex (G4) forming sequences in telomeric DNA and c-myc promoter regions of human DNA are associated with tumorogenesis. Ligands that can facilitate or stabilize the formation and increase the stabilization of G4 can prevent tumor cell proliferation and have been regarded as potential anti-cancer drugs. In the present study, steady state and time-resolved fluorescence measurements provide important structural and dynamical insights into the free and bound states of the therapeutically potent plant flavonoid fisetin (3,3′,4′,7-tetrahydroxyflavone) in a G4 DNA matrix. The excited state intra-molecular proton transfer (ESPT) of fisetin plays an important role in observing and understanding the binding of fisetin with the G4 DNA. Differential absorption spectra, thermal melting, and circular dichroism spectroscopic studies provide evidences for the formation of G4 DNA and size exclusion chromatography (SEC) proves the binding and 1∶1 stoichiometry of fisetin in the DNA matrix. Comparative analysis of binding in the presence of EtBr proves that fisetin favors binding at the face of the G-quartet, mostly along the diagonal loop. Time resolved fluorescence anisotropy decay analysis indicates the increase in the restrictions in motion from the free to bound fisetin. We have also investigated the fingerprints of the binding of fisetin in the antiparallel quadruplex using Raman spectroscopy. Preliminary results indicate fisetin to be a prospective candidate as a G4 ligand. PMID:23785423

  2. Modulation of Intersystem Crossing Rate by Minor Ligand Modifications in Cyclometalated Platinum(II) Complexes.

    PubMed

    Shafikov, Marsel Z; Kozhevnikov, Dmitry N; Bodensteiner, Michael; Brandl, Fabian; Czerwieniec, Rafał

    2016-08-01

    Photophysical properties of four new platinum(II) complexes comprising extended ppy (Hppy = 2-phenylpyridine) and thpy (Hthpy = 2-(2'-thienyl)pyridine) cyclometalated ligands and acetylacetonate (acac) are reported. Substitution of the benzene ring of Pt-ppy complexes 1 and 2 with a more electron-rich thiophene of Pt-thpy complexes 3 and 4 leads to narrowing of the HOMO-LUMO gap and thus to a red shift of the lowest energy absorption band and phosphorescence band, as expected for low-energy excited states of the intraligand/metal-to-ligand charge transfer character. However, in addition to these conventional spectral shifts, another, at first unexpected, substitution effect occurs. Pt-thpy complexes 3 and 4 are dual emissive showing fluorescence about 6000 cm(-1) (∼0.75 eV) higher in energy relative to the phosphorescence band, while for Pt-ppy complexes 1 and 2 only phosphorescence is observed. For dual-emissive complexes 3 and 4, ISC rates kISC are estimated to be in order of 10(9)-10(10) s(-1), while kISC of Pt-ppy complexes 1 and 2 is much faster amounting to 10(12) s(-1) or more. The relative intensities of the fluorescence and phosphorescence signals of Pt-thpy complexes 3 and 4 depend on the excitation wavelength, showing that hyper-intersystem crossing (HISC) in these complexes is observably significant. PMID:27388146

  3. Synthesis and Characterisation of Copper(II) Complexes with Tridentate NNO Functionalized Ligand: Density Function Theory Study, DNA Binding Mechanism, Optical Properties, and Biological Application

    PubMed Central

    Hazra, Madhumita; Dolai, Tanushree; Pandey, Akhil; Dey, Subrata Kumar; Patra, Animesh

    2014-01-01

    The photo physical properties of two mononuclear pentacoordinated copper(II) complexes formulated as [Cu(L)(Cl)(H2O)] (1) and [Cu(L)(Br)(H2O)] (2) HL = (1-[(3-methyl-pyridine-2-ylimino)-methyl]-naphthalen-2-ol) were synthesized and characterized by elemental, physicochemical, and spectroscopic methods. The density function theory calculations are used to investigate the electronic structures and the electronic properties of ligand and complex. The interactions of copper(II) complexes towards calf thymus DNA were examined with the help of absorption, viscosity, and fluorescence spectroscopic techniques at pH 7.40. All spectroscopy's result indicates that complexes show good binding activity to calf thymus DNA through groove binding. The optical absorption and fluorescence emission properties of microwires were characterized by fluorescence microscope. From a spectroscopic viewpoint, all compounds strongly emit green light in the solid state. The microscopy investigation suggested that microwires exhibited optical waveguide behaviour which are applicable as fluorescent nanomaterials and can be used as building blocks for miniaturized photonic devices. Antibacterial study reveals that complexes are better antimicrobial agents than free Schiff base due to bacterial cell penetration by chelation. Moreover, the antioxidant study of the ligand and complexes is evaluated by using 1,1-diphenyl-2-picrylhydrazyl (DPPH) free-radical assays, which demonstrate that the complexes are of higher antioxidant activity than free ligand. PMID:25386109

  4. Shedding Some Light on Fluorescent Bulbs.

    ERIC Educational Resources Information Center

    Guilbert, Nicholas R.

    1996-01-01

    Explores some of the principles behind the working of fluorescent bulbs using a specially prepared fluorescent bulb with the white inner fluorescent coating applied along only half its length. Discusses the spectrum, the bulb plasma, and light production. (JRH)

  5. Ammonia formation by metal-ligand cooperative hydrogenolysis of a nitrido ligand

    NASA Astrophysics Data System (ADS)

    Askevold, Bjorn; Nieto, Jorge Torres; Tussupbayev, Samat; Diefenbach, Martin; Herdtweck, Eberhardt; Holthausen, Max C.; Schneider, Sven

    2011-07-01

    Bioinspired hydrogenation of N2 to ammonia at ambient conditions by stepwise nitrogen protonation/reduction with metal complexes in solution has experienced remarkable progress. In contrast, the highly desirable direct hydrogenation with H2 remains difficult. In analogy to the heterogeneously catalysed Haber-Bosch process, such a reaction is conceivable via metal-centred N2 splitting and unprecedented hydrogenolysis of the nitrido ligands to ammonia. We report the synthesis of a ruthenium(IV) nitrido complex. The high nucleophilicity of the nitrido ligand is demonstrated by unusual N-C coupling with π-acidic CO. Furthermore, the terminal nitrido ligand undergoes facile hydrogenolysis with H2 at ambient conditions to produce ammonia in high yield. Kinetic and quantum chemical examinations of this reaction suggest cooperative behaviour of a phosphorus-nitrogen-phosphorus pincer ligand in rate-determining heterolytic hydrogen splitting.

  6. Ammonia formation by metal-ligand cooperative hydrogenolysis of a nitrido ligand.

    PubMed

    Askevold, Bjorn; Nieto, Jorge Torres; Tussupbayev, Samat; Diefenbach, Martin; Herdtweck, Eberhardt; Holthausen, Max C; Schneider, Sven

    2011-07-01

    Bioinspired hydrogenation of N(2) to ammonia at ambient conditions by stepwise nitrogen protonation/reduction with metal complexes in solution has experienced remarkable progress. In contrast, the highly desirable direct hydrogenation with H(2) remains difficult. In analogy to the heterogeneously catalysed Haber-Bosch process, such a reaction is conceivable via metal-centred N(2) splitting and unprecedented hydrogenolysis of the nitrido ligands to ammonia. We report the synthesis of a ruthenium(IV) nitrido complex. The high nucleophilicity of the nitrido ligand is demonstrated by unusual N-C coupling with π-acidic CO. Furthermore, the terminal nitrido ligand undergoes facile hydrogenolysis with H(2) at ambient conditions to produce ammonia in high yield. Kinetic and quantum chemical examinations of this reaction suggest cooperative behaviour of a phosphorus-nitrogen-phosphorus pincer ligand in rate-determining heterolytic hydrogen splitting. PMID:21697873

  7. Mixed-ligand copper(ii) Schiff base complexes: the role of the co-ligand in DNA binding, DNA cleavage, protein binding and cytotoxicity.

    PubMed

    Lian, Wen-Jing; Wang, Xin-Tian; Xie, Cheng-Zhi; Tian, He; Song, Xue-Qing; Pan, He-Ting; Qiao, Xin; Xu, Jing-Yuan

    2016-05-31

    Four novel mononuclear Schiff base copper(ii) complexes, namely, [Cu(L)(OAc)]·H2O (), [Cu(HL)(C2O4)(EtOH)]·EtOH (), [Cu(L)(Bza)] () and [Cu(L)(Sal)] () (HL = 1-(((2-((2-hydroxypropyl)amino)ethyl)imino)methyl)naphthalene-2-ol), Bza = benzoic acid, Sal = salicylic acid), were synthesized and characterized by X-ray crystallography, elemental analysis and infrared spectroscopy. Single-crystal diffraction analysis revealed that all the complexes were mononuclear molecules, in which the Schiff base ligand exhibited different coordination modes and conformations. The N-HO and O-HO inter- and intramolecular hydrogen bonding interactions linked these molecules into multidimensional networks. Their interactions with calf thymus DNA (CT-DNA) were investigated by UV-visible and fluorescence spectrometry, as well as by viscosity measurements. The magnitude of the Kapp values of the four complexes was 10(5), indicating a moderate intercalative binding mode between the complexes and DNA. Electrophoresis results showed that all these complexes induced double strand breaks of pUC19 plasmid DNA in the presence of H2O2 through an oxidative pathway. In addition, the fluorescence spectrum of human serum albumin (HSA) with the complexes suggested that the quenching mechanism of HSA by the complexes was a static process. Moreover, the antiproliferative activity of the four complexes against HeLa (human cervical carcinoma) and HepG-2 (human liver hepatocellular carcinoma) cells evaluated by colorimetric cell proliferation assay and clonogenic assay revealed that all four complexes had improved cytotoxicity against cancer cells. Inspiringly, complex , with salicylic acid as the auxiliary ligand, displayed a stronger anticancer activity, suggesting that a synergistic effect of the Schiff base complex and the nonsteroidal anti-inflammatory drug may be involved in the cell killing process. The biological features of mixed-ligand copper(ii) Schiff base complexes and how acetic auxiliary

  8. On the Design of Low-Cost Fluorescent Protein Biosensors

    NASA Astrophysics Data System (ADS)

    Tolosa, Leah

    Proteins are the cell’s workhorses as they are involved in essentially all cellular activities. From the birth of a daughter cell until its death during apoptosis, proteins perform various functions as needed. Such versatility can be attributed to the seemingly infinite ways that the amino acids are sequenced in a polypeptide. The polypeptide backbone and each amino acid functional group contribute unique intermolecular interactions that determine the protein’s shape and size. However, more importantly, these interactions determine protein function, that is, its ability to interact with the external environment. For the same reasons, proteins make ideal biorecognition elements for sensors (see below and Fig. 1). Protein enzymes and receptors developed unmatched sensitivity and selectivity for their substrates through millions of years of natural selection. In contrast, chemically synthesized artificial receptors seldom approach the same level of sophistication in ligand selectivity, sensitivity range and ease of production. In addition, there is available to the sensor researcher an ever expanding library of proteins for various ligands important in all manner of application such as disease biomarkers, toxins, contaminants, drugs, etc. Using tools of genetic engineering, researchers can even go beyond known existing wild type proteins by introducing useful functional groups thereby tuning or even altering protein sensitivity and selectivity. This can be done by mutagenesis and directed evolution or by incorporation of unnatural amino acids during translation. Additionally, genes for the protein of interest can be inserted in vectors predesigned with handles for affinity purification or immobilization on a surface. Recombinant proteins can then be produced in large amounts in appropriate cellular hosts with very high reproducibility. In addition, chemical reactions involving the amino acids have become standard protocols. Thus, there are thousands of dyes

  9. All-inorganic Germanium nanocrystal films by cationic ligand exchange

    DOE PAGESBeta

    Wheeler, Lance M.; Nichols, Asa W.; Chernomordik, Boris D.; Anderson, Nicholas C.; Beard, Matthew C.; Neale, Nathan R.

    2016-01-21

    In this study, we introduce a new paradigm for group IV nanocrystal surface chemistry based on room temperature surface activation that enables ionic ligand exchange. Germanium nanocrystals synthesized in a gas-phase plasma reactor are functionalized with labile, cationic alkylammonium ligands rather than with traditional covalently bound groups. We employ Fourier transform infrared and 1H nuclear magnetic resonance spectroscopies to demonstrate the alkylammonium ligands are freely exchanged on the germanium nanocrystal surface with a variety of cationic ligands, including short inorganic ligands such as ammonium and alkali metal cations. This ionic ligand exchange chemistry is used to demonstrate enhanced transport inmore » germanium nanocrystal films following ligand exchange as well as the first photovoltaic device based on an all-inorganic germanium nanocrystal absorber layer cast from solution. This new ligand chemistry should accelerate progress in utilizing germanium and other group IV nanocrystals for optoelectronic applications.« less

  10. All-Inorganic Germanium Nanocrystal Films by Cationic Ligand Exchange.

    PubMed

    Wheeler, Lance M; Nichols, Asa W; Chernomordik, Boris D; Anderson, Nicholas C; Beard, Matthew C; Neale, Nathan R

    2016-03-01

    We introduce a new paradigm for group IV nanocrystal surface chemistry based on room temperature surface activation that enables ionic ligand exchange. Germanium nanocrystals synthesized in a gas-phase plasma reactor are functionalized with labile, cationic alkylammonium ligands rather than with traditional covalently bound groups. We employ Fourier transform infrared and (1)H nuclear magnetic resonance spectroscopies to demonstrate the alkylammonium ligands are freely exchanged on the germanium nanocrystal surface with a variety of cationic ligands, including short inorganic ligands such as ammonium and alkali metal cations. This ionic ligand exchange chemistry is used to demonstrate enhanced transport in germanium nanocrystal films following ligand exchange as well as the first photovoltaic device based on an all-inorganic germanium nanocrystal absorber layer cast from solution. This new ligand chemistry should accelerate progress in utilizing germanium and other group IV nanocrystals for optoelectronic applications. PMID:26796765

  11. Quantum.Ligand.Dock: protein-ligand docking with quantum entanglement refinement on a GPU system.

    PubMed

    Kantardjiev, Alexander A

    2012-07-01

    Quantum.Ligand.Dock (protein-ligand docking with graphic processing unit (GPU) quantum entanglement refinement on a GPU system) is an original modern method for in silico prediction of protein-ligand interactions via high-performance docking code. The main flavour of our approach is a combination of fast search with a special account for overlooked physical interactions. On the one hand, we take care of self-consistency and proton equilibria mutual effects of docking partners. On the other hand, Quantum.Ligand.Dock is the the only docking server offering such a subtle supplement to protein docking algorithms as quantum entanglement contributions. The motivation for development and proposition of the method to the community hinges upon two arguments-the fundamental importance of quantum entanglement contribution in molecular interaction and the realistic possibility to implement it by the availability of supercomputing power. The implementation of sophisticated quantum methods is made possible by parallelization at several bottlenecks on a GPU supercomputer. The high-performance implementation will be of use for large-scale virtual screening projects, structural bioinformatics, systems biology and fundamental research in understanding protein-ligand recognition. The design of the interface is focused on feasibility and ease of use. Protein and ligand molecule structures are supposed to be submitted as atomic coordinate files in PDB format. A customization section is offered for addition of user-specified charges, extra ionogenic groups with intrinsic pK(a) values or fixed ions. Final predicted complexes are ranked according to obtained scores and provided in PDB format as well as interactive visualization in a molecular viewer. Quantum.Ligand.Dock server can be accessed at http://87.116.85.141/LigandDock.html. PMID:22669908

  12. Resonance energy transfer-amplifying fluorescence quenching at the surface of silica nanoparticles toward ultrasensitive detection of TNT.

    PubMed

    Gao, Daming; Wang, Zhenyang; Liu, Bianhua; Ni, Lin; Wu, Minghong; Zhang, Zhongping

    2008-11-15

    This paper reports a resonance energy transfer-amplifying fluorescence quenching at the surface of silica nanoparticles for the ultrasensitive detection of 2,4,6-trinitrotoluene (TNT) in solution and vapor environments. Fluorescence dye and organic amine were covalently modified onto the surface of silica nanoparticles to form a hybrid monolayer of dye fluorophores and amine ligands. The fluorescent silica particles can specifically bind TNT species by the charge-transfer complexing interaction between electron-rich amine ligands and electron-deficient aromatic rings. The resultant TNT-amine complexes bound at the silica surface can strongly suppress the fluorescence emission of the chosen dye by the fluorescence resonance energy transfer (FRET) from dye donor to the irradiative TNT-amine acceptor through intermolecular polar-polar interactions at spatial proximity. The quenching efficiency of the hybrid nanoparticles with TNT is greatly amplified by at least 10-fold that of the corresponding pure dye. The nanoparticle-assembled arrays on silicon wafer can sensitively detect down to approximately 1 nM TNT with the use of only 10 microL of solution (approximately 2 pg TNT) and several ppb of TNT vapor in air. The simple FRET-based nanoparticle sensors reported here exhibit a high and stable fluorescence brightness, strong analyte affinity, and good assembly flexibility and can thus find many applications in the detection of ultratrace analytes. PMID:18847285

  13. Ligand-dependent localization and function of ORP-VAP complexes at membrane contact sites.

    PubMed

    Weber-Boyvat, Marion; Kentala, Henriikka; Peränen, Johan; Olkkonen, Vesa M

    2015-05-01

    Oxysterol-binding protein/OSBP-related proteins (ORPs) constitute a conserved family of sterol/phospholipid-binding proteins with lipid transporter or sensor functions. We investigated the spatial occurrence and regulation of the interactions of human OSBP/ORPs or the S. cerevisiae orthologs, the Osh (OSBP homolog) proteins, with their endoplasmic reticulum (ER) anchors, the VAMP-associated proteins (VAPs), by employing bimolecular fluorescence complementation and pull-down set-ups. The ORP-VAP interactions localize frequently at distinct subcellular sites, shown in several cases to represent membrane contact sites (MCSs). Using established ORP ligand-binding domain mutants and pull-down assays with recombinant proteins, we show that ORP liganding regulates the ORP-VAP association, alters the subcellular targeting of ORP-VAP complexes, or modifies organelle morphology. There is distinct protein specificity in the effects of the mutants on subcellular targeting of ORP-VAP complexes. We provide evidence that complexes of human ORP2 and VAPs at ER-lipid droplet interfaces regulate the hydrolysis of triglycerides and lipid droplet turnover. The data suggest evolutionarily conserved, complex ligand-dependent functions of ORP-VAP complexes at MCSs, with implications for cellular lipid homeostasis and signaling. PMID:25420878

  14. Improving Binding Affinity and Selectivity of Computationally Designed Ligand-Binding Proteins Using Experiments.

    PubMed

    Tinberg, Christine E; Khare, Sagar D

    2016-01-01

    The ability to de novo design proteins that can bind small molecules has wide implications for synthetic biology and medicine. Combining computational protein design with the high-throughput screening of mutagenic libraries of computationally designed proteins is emerging as a general approach for creating binding proteins with programmable binding modes, affinities, and selectivities. The computational step enables the creation of a binding site in a protein that otherwise does not (measurably) bind the intended ligand, and targeted mutagenic screening allows for validation and refinement of the computational model as well as provides orders-of-magnitude increases in the binding affinity. Deep sequencing of mutagenic libraries can provide insights into the mutagenic binding landscape and enable further affinity improvements. Moreover, in such a combined computational-experimental approach where the binding mode is preprogrammed and iteratively refined, selectivity can be achieved (and modulated) by the placement of specified amino acid side chain groups around the ligand in defined orientations. Here, we describe the experimental aspects of a combined computational-experimental approach for designing-using the software suite Rosetta-proteins that bind a small molecule of choice and engineering, using fluorescence-activated cell sorting and high-throughput yeast surface display, high affinity and ligand selectivity. We illustrated the utility of this approach by performing the design of a selective digoxigenin (DIG)-binding protein that, after affinity maturation, binds DIG with picomolar affinity and high selectivity over structurally related steroids. PMID:27094290

  15. Targeted delivery of anti-coxsackievirus siRNAs using ligand-conjugated packaging RNAs.

    PubMed

    Zhang, Huifang M; Su, Yue; Guo, Songchuan; Yuan, Ji; Lim, Travis; Liu, Jing; Guo, Peixuan; Yang, Decheng

    2009-09-01

    Coxsackievirus B3 (CVB3) is a common pathogen of myocarditis. We previously synthesized a siRNA targeting the CVB3 protease 2A (siRNA/2A) gene and achieved reduction of CVB3 replication by 92% in vitro. However, like other drugs under development, CVB3 siRNA faces a major challenge of targeted delivery. In this study, we investigated a novel approach to deliver CVB3 siRNAs to a specific cell population (e.g. HeLa cells containing folate receptor) using receptor ligand (folate)-linked packaging RNA (pRNA) from bacterial phage phi29. pRNA monomers can spontaneously form dimers and multimers under optimal conditions by base-pairing between their stem loops. By covalently linking a fluorescence-tag to folate, we delivered the conjugate specifically to HeLa cells without the need of transfection. We further demonstrated that pRNA covalently conjugated to siRNA/2A achieved an equivalent antiviral effect to that of the siRNA/2A alone. Finally, the drug targeted delivery was further evaluated by using pRNA monomers or dimers, which carried both the siRNA/2A and folate ligand and demonstrated that both of them strongly inhibited CVB3 replication. These data indicate that pRNA as a siRNA carrier can specifically deliver the drug to target cells via its ligand and specific receptor interaction and inhibit virus replication effectively. PMID:19616030

  16. Near-IR luminescent lanthanide complexes with 1,8-diaminoanthraquinone-based chromophoric ligands.

    PubMed

    Stacey, Oliver J; Ward, Benjamin D; Amoroso, Angelo J; Pope, Simon J A

    2016-04-12

    Three new chromophoric anthraquinone-based multidentate ligands have been synthesised in a step-wise manner from 1,8-dichloroanthraquinone. The ligands each comprise two dipicolyl amine units and react with trivalent lanthanide ions to form monometallic complexes of the form [Ln(L)](OTf)3 as indicated by MS studies and elemental analyses. Supporting DFT studies show that the monometallic species are highly favoured (>1000 kJ mol(-1)) over the formation of a 2 : 2 dimetallic congener. Both ligands and complexes absorb light efficiently (ε ∼ 10(4) M(-1) cm(-1)) in the visible part of the spectrum, with λabsca. 535-550 nm through an intramolecular charge transfer (ICT) transition localised on the substituted anthraquinone unit. In all cases the complexes show a fluorescence band at ca. 675 nm due to the ICT emitting state. The corresponding Nd(iii), Yb(iii) and Er(iii) complexes also reveal sensitised near-IR emission characteristic of each ion following excitation of the ICT visible absorption band at 535 nm. PMID:26974664

  17. Domain Architecture of a Calcium-Permeable AMPA Receptor in a Ligand-Free Conformation

    PubMed Central

    Midgett, Charles R.; Gill, Avinash; Madden, Dean R.

    2012-01-01

    Ligand-gated ion channels couple the free energy of agonist binding to the gating of selective transmembrane ion pores, permitting cells to regulate ion flux in response to external chemical stimuli. However, the stereochemical mechanisms responsible for this coupling remain obscure. In the case of the ionotropic glutamate receptors (iGluRs), the modular nature of receptor subunits has facilitated structural analysis of the N-terminal domain (NTD), and of multiple conformations of the ligand-binding domain (LBD). Recently, the crystallographic structure of an antagonist-bound form of the receptor was determined. However, disulfide trapping of this conformation blocks channel opening, suggesting that channel activation involves additional quaternary packing arrangements. To explore the conformational space available to iGluR channels, we report here a second, clearly distinct domain architecture of homotetrameric, calcium-permeable AMPA receptors, determined by single-particle electron microscopy of untagged and fluorescently tagged constructs in a ligand-free state. It reveals a novel packing of NTD dimers, and a separation of LBD dimers across a central vestibule. In this arrangement, which reconciles diverse functional observations, agonist-induced cleft closure across LBD dimers can be converted into a twisting motion that provides a basis for receptor activation. PMID:22232575

  18. Influence of neutral amine ligands on the network assembly of lead(II) 4-sulfobenzoate complexes

    NASA Astrophysics Data System (ADS)

    Zhang, Li-Ping; Zhu, Long-Guan

    2008-02-01

    Two new lead(II) 4-sulfobenzoate complexes with amine ligands, [Pb(4-sb)(2,2'-bipy)] n ( 1) and {[Pb(4-sb)(4,4'-bipy) 1/2] · (4,4'-bipy) 1/2} n ( 2) (2,2'-bipy = 2,2'-bipyridine; 4,4'-bipy = 4,4'-bipyridine; 4-sb = 4-sulfobenzoate dianion), were synthesized and characterized by single-crystal X-ray analyses, IR, TG, elemental analyses and fluorescent studies. Complexes 1 and 2, in addition to previously reported [Pb(4-sb)(H 2O) 2] n ( 3) and {[Pb(4-sb)(phen)] · (H 2O)} n ( 4) where phen is 1,10-phenanthroline, all contain [Pb(4-sb)] building units, while four [Pb(4-sb)] networks in 1- 4 are very different, which is significantly influenced by the neutral ligands. The coordination array of complex 1 is a 2-D network in which each 4-sb acts as a η 5-μ 3 mode while the complex 2 is a 3-D architecture in which each 4-sb performs a η 5-μ 6 mode. Both coordination modes of 4-sb ligands in 1- 2 are novel and first reported in this paper.

  19. Non-Ligand-Induced Dimerization is Sufficient to Initiate the Signalling and Endocytosis of EGF Receptor

    PubMed Central

    Kourouniotis, George; Wang, Yi; Pennock, Steven; Chen, Xinmei; Wang, Zhixiang

    2016-01-01

    The binding of epidermal growth factor (EGF) to EGF receptor (EGFR) stimulates cell mitogenesis and survival through various signalling cascades. EGF also stimulates rapid EGFR endocytosis and its eventual degradation in lysosomes. The immediate events induced by ligand binding include receptor dimerization, activation of intrinsic tyrosine kinase and autophosphorylation. However, in spite of intensified efforts, the results regarding the roles of these events in EGFR signalling and internalization is still very controversial. In this study, we constructed a chimeric EGFR by replacing its extracellular domain with leucine zipper (LZ) and tagged a green fluorescent protein (GFP) at its C-terminus. We showed that the chimeric LZ-EGFR-GFP was constitutively dimerized. The LZ-EGFR-GFP dimer autophosphorylated each of its five well-defined C-terminal tyrosine residues as the ligand-induced EGFR dimer does. Phosphorylated LZ-EGFR-GFP was localized to both the plasma membrane and endosomes, suggesting it is capable of endocytosis. We also showed that LZ-EGFR-GFP activated major signalling proteins including Src homology collagen-like (Shc), extracellular signal-regulated kinase (ERK) and Akt. Moreover, LZ-EGFR-GFP was able to stimulate cell proliferation. These results indicate that non-ligand induced dimerization is sufficient to activate EGFR and initiate cell signalling and EGFR endocytosis. We conclude that receptor dimerization is a critical event in EGF-induced cell signalling and EGFR endocytosis. PMID:27463710

  20. Affinity thermoprecipitatin: Contribution of the efficiency of ligand-protein interaction and access of the ligand.

    PubMed

    Galaev, I Y; Mattiasson, B

    1993-05-01

    Conjugates to two thermoprecipitating polymers, poly(N-vinyl caprolactam) and poly(N-isopropylacrylmide), with soybean trypsin inhibitor, Cibacron Blue 3GA, Cu-iminodiacetic acid, and p-aminobenzamidine were synthesized. The interaction of these conjugates with trypsin and lactate dehydrogenase was studied. Coupling of the ligand to a polymer resulted in a 100-1000-fold decrease in enzyme-affinity. Rough theoretical estimates revealed that a successful affinity precipitation required that the binding of a target protein and a ligand coupled to a polymer have binding constants on the order of 10(-7)-10(-8) M. Such strong affinity of low molecular weight ligands that can provide binding constants of 10(-9)-10(-11) M or alternatively multipoint attachment of the target protein molecule. The ligand in the ligand-polymer conjugate is still accessible to the protein after thermoprecipitation, and the latter can bind with the particle of the dispersion of thermoprecipitated ligand-polymer precipitate may result in stripping of enzyme molecules from the surface of the particles. PMID:18601296