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116 Publications

Showing 91-100 of 116 results
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    01/28/14 | Single-molecule tracking of the transcription cycle by sub-second RNA detection.
    Zhang Z, Revyakin A, Grimm JB, Lavis LD, Tjian R
    eLife. 2014 Jan 28;3:e01775. doi: 10.7554/eLife.01775

    Transcription is an inherently stochastic, noisy, and multi-step process, in which fluctuations at every step can cause variations in RNA synthesis, and affect physiology and differentiation decisions in otherwise identical cells. However, it has been an experimental challenge to directly link the stochastic events at the promoter to transcript production. Here we established a fast fluorescence in situ hybridization (fastFISH) method that takes advantage of intrinsically unstructured nucleic acid sequences to achieve exceptionally fast rates of specific hybridization (\~{}10e7 M(-1)s(-1)), and allows deterministic detection of single nascent transcripts. Using a prototypical RNA polymerase, we demonstrated the use of fastFISH to measure the kinetic rates of promoter escape, elongation, and termination in one assay at the single-molecule level, at sub-second temporal resolution. The principles of fastFISH design can be used to study stochasticity in gene regulation, to select targets for gene silencing, and to design nucleic acid nanostructures. DOI: http://dx.doi.org/10.7554/eLife.01775.001.

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    11/01/13 | Caged naloxone reveals opioid signaling deactivation kinetics.
    Banghart MR, Williams JT, Shah RC, Lavis LD, Sabatini BL
    Molecular Pharmacology. 2013 Nov;84(5):687-95. doi: 10.1124/mol.113.088096

    The spatiotemporal dynamics of opioid signaling in the brain remain poorly defined. Photoactivatable opioid ligands provide a means to quantitatively measure these dynamics and their underlying mechanisms in brain tissue. Although activation kinetics can be assessed using caged agonists, deactivation kinetics are obscured by slow clearance of agonist in tissue. To reveal deactivation kinetics of opioid signaling we developed a caged competitive antagonist that can be quickly photoreleased in sufficient concentrations to render agonist dissociation effectively irreversible. Carboxynitroveratryl-naloxone (CNV-NLX), a caged analog of the competitive opioid antagonist NLX, was readily synthesized from commercially available NLX in good yield and found to be devoid of antagonist activity at heterologously expressed opioid receptors. Photolysis in slices of rat locus coeruleus produced a rapid inhibition of the ionic currents evoked by multiple agonists of the μ-opioid receptor (MOR), but not of α-adrenergic receptors, which activate the same pool of ion channels. Using the high-affinity peptide agonist dermorphin, we established conditions under which light-driven deactivation rates are independent of agonist concentration and thus intrinsic to the agonist-receptor complex. Under these conditions, some MOR agonists yielded deactivation rates that are limited by G protein signaling, whereas others appeared limited by agonist dissociation. Therefore, the choice of agonist determines which feature of receptor signaling is unmasked by CNV-NLX photolysis.

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    06/17/13 | Decoupled roles for the atypical, bifurcated binding pocket of the ybfF hydrolase.
    Ellis EE, Adkins CT, Galovska NM, Lavis LD, Johnson RJ
    Chembiochem : A European Journal of Chemical Biology. 2013 Jun 17;14(9):1134-44. doi: 10.1002/cbic.201300085

    Serine hydrolases have diverse intracellular substrates, biological functions, and structural plasticity, and are thus important for biocatalyst design. Amongst serine hydrolases, the recently described ybfF enzyme family are promising novel biocatalysts with an unusual bifurcated substrate-binding cleft and the ability to recognize commercially relevant substrates. We characterized in detail the substrate selectivity of a novel ybfF enzyme from Vibrio cholerae (Vc-ybfF) by using a 21-member library of fluorogenic ester substrates. We assigned the roles of the two substrate-binding clefts in controlling the substrate selectivity and folded stability of Vc-ybfF by comprehensive substitution analysis. The overall substrate preference of Vc-ybfF was for short polar chains, but it retained significant activity with a range of cyclic and extended esters. This broad substrate specificity combined with the substitutional analysis demonstrates that the larger binding cleft controls the substrate specificity of Vc-ybfF. Key selectivity residues (Tyr116, Arg120, Tyr209) are also located at the larger binding pocket and control the substrate specificity profile. In the structure of ybfF the narrower binding cleft contains water molecules prepositioned for hydrolysis, but based on substitution this cleft showed only minimal contribution to catalysis. Instead, the residues surrounding the narrow binding cleft and at the entrance to the binding pocket contributed significantly to the folded stability of Vc-ybfF. The relative contributions of each cleft of the binding pocket to the catalytic activity and folded stability of Vc-ybfF provide a valuable map for designing future biocatalysts based on the ybfF scaffold.

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    04/24/13 | Carbofluoresceins and carborhodamines as scaffolds for high-contrast fluorogenic probes.
    Grimm JB, Sung AJ, Legant WR, Hulamm P, Matlosz SM, Betzig E, Lavis LD
    ACS Chemical Biology. 2013 Apr 24;8(6):1303-10. doi: 10.1021/cb4000822

    Fluorogenic molecules are important tools for advanced biochemical and biological experiments. The extant collection of fluorogenic probes is incomplete, however, leaving regions of the electromagnetic spectrum unutilized. Here, we synthesize green-excited fluorescent and fluorogenic analogues of the classic fluorescein and rhodamine 110 fluorophores by replacement of the xanthene oxygen with a quaternary carbon. These anthracenyl "carbofluorescein" and "carborhodamine 110" fluorophores exhibit excellent fluorescent properties and can be masked with enzyme- and photolabile groups to prepare high-contrast fluorogenic molecules useful for live cell imaging experiments and super-resolution microscopy. Our divergent approach to these red-shifted dye scaffolds will enable the preparation of numerous novel fluorogenic probes with high biological utility.

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    01/01/13 | The chemistry of small-molecule fluorogenic probes.
    Grimm JB, Heckman LM, Lavis LD
    Progress in Molecular Biology and Translational Science;113:1-34. doi: 10.1016/B978-0-12-386932-6.00001-6

    Chemical fluorophores find wide use in biology to detect and visualize different phenomena. A key advantage of small-molecule dyes is the ability to construct compounds where fluorescence is activated by chemical or biochemical processes. Fluorogenic molecules, in which fluorescence is activated by enzymatic activity, light, or environmental changes, enable advanced bioassays and sophisticated imaging experiments. Here, we detail the collection of fluorophores and highlight both general strategies and unique approaches that are employed to control fluorescence using chemistry.

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    09/01/12 | The structural basis for the narrow substrate specificity of an acetyl esterase from Thermotoga maritima.
    Hedge MK, Gehring AM, Adkins CT, Weston LA, Lavis LD, Johnson RJ
    Biochimica et Biophysica Acta. 2012 Sep;1824(9):1024-30. doi: 10.1016/j.bbapap.2012.05.009

    Acetyl esterases from carbohydrate esterase family 7 exhibit unusual substrate specificity. These proteins catalyze the cleavage of disparate acetate esters with high efficiency, but are unreactive to larger acyl groups. The structural basis for this distinct selectivity profile is unknown. Here, we investigate a thermostable acetyl esterase (TM0077) from Thermotoga maritima using evolutionary relationships, structural information, fluorescent kinetic measurements, and site directed mutagenesis. We measured the kinetic and structural determinants for this specificity using a diverse series of small molecule enzyme substrates, including novel fluorogenic esters. These experiments identified two hydrophobic plasticity residues (Pro228, and Ile276) surrounding the nucleophilic serine that impart this specificity of TM0077 for small, straight-chain esters. Substitution of these residues with alanine imparts broader specificity to TM0077 for the hydrolysis of longer and bulkier esters. Our results suggest the specificity of acetyl esterases have been finely tuned by evolution to catalyze the removal of acetate groups from diverse substrates, but can be modified by focused amino acid substitutions to yield enzymes capable of cleaving larger ester functionalities.

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    Looger LabLavis Lab
    04/01/12 | A genetically encoded fluorescent protein in echinoderms marks the history of neuronal activity.
    Verdecia MA, Looger LL, Lavis L, Graumann J, Mandel G, Brehm P
    Luminescence. 2012 Apr;27:170

    Since the original identification of GFP from jellyfish and corals, the genetically encoded fluorescent proteins have become mainstream indicators for imaging. Functionally homologous candidates exist in more highly evolved bioluminescent invertebrates, including echinoderms. For example, in brittlestars, stimulus-evoked bioluminescence is transient, lasting seconds, and emanates from specialized cells (photocytes). Prior to light emission, we observe little or no green fluorescence. However, concurrent with light emission, an intense green, calcium-dependent fluorescence develops that persists indefinitely.

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    Sternson LabLooger LabLavis Lab
    03/27/12 | Selective esterase-ester pair for targeting small molecules with cellular specificity.
    Tian L, Yang Y, Wysocki LM, Arnold AC, Hu A, Ravichandran B, Sternson SM, Looger LL, Lavis LD
    Proceedings of the National Academy of Sciences of the United States of America. 2012 Mar 27;109:4756-61. doi: 10.1073/pnas.1111943109

    Small molecules are important tools to measure and modulate intracellular signaling pathways. A longstanding limitation for using chemical compounds in complex tissues has been the inability to target bioactive small molecules to a specific cell class. Here, we describe a generalizable esterase-ester pair capable of targeted delivery of small molecules to living cells and tissue with cellular specificity. We used fluorogenic molecules to rapidly identify a small ester masking motif that is stable to endogenous esterases, but is efficiently removed by an exogenous esterase. This strategy allows facile targeting of dyes and drugs in complex biological environments to label specific cell types, illuminate gap junction connectivity, and pharmacologically perturb distinct subsets of cells. We expect this approach to have general utility for the specific delivery of many small molecules to defined cellular populations.

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    02/22/12 | Excitation spectra and brightness optimization of two-photon excited probes.
    Mütze J, Iyer V, Macklin JJ, Colonell J, Karsh B, Petrá\v sek Ze, Schwille P, Looger LL, Lavis LD, Harris TD
    Biophysical Journal. 2012 Feb 22;102(4):934-44. doi: 10.1016/j.bpj.2011.12.056

    Two-photon probe excitation data are commonly presented as absorption cross section or molecular brightness (the detected fluorescence rate per molecule). We report two-photon molecular brightness spectra for a diverse set of organic and genetically encoded probes with an automated spectroscopic system based on fluorescence correlation spectroscopy. The two-photon action cross section can be extracted from molecular brightness measurements at low excitation intensities, while peak molecular brightness (the maximum molecular brightness with increasing excitation intensity) is measured at higher intensities at which probe photophysical effects become significant. The spectral shape of these two parameters was similar across all dye families tested. Peak molecular brightness spectra, which can be obtained rapidly and with reduced experimental complexity, can thus serve as a first-order approximation to cross-section spectra in determining optimal wavelengths for two-photon excitation, while providing additional information pertaining to probe photostability. The data shown should assist in probe choice and experimental design for multiphoton microscopy studies. Further, we show that, by the addition of a passive pulse splitter, nonlinear bleaching can be reduced-resulting in an enhancement of the fluorescence signal in fluorescence correlation spectroscopy by a factor of two. This increase in fluorescence signal, together with the observed resemblance of action cross section and peak brightness spectra, suggests higher-order photobleaching pathways for two-photon excitation.

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    12/16/11 | Synthesis of rhodamines from fluoresceins using Pd-catalyzed C-N cross-coupling.
    Grimm JB, Lavis LD
    Organic Letters. 2011 Dec 16;13(24):6354-7. doi: 10.1021/ol202618t

    A unified, convenient, and efficient strategy for the preparation of rhodamines and N,N’-diacylated rhodamines has been developed. Fluorescein ditriflates were found to undergo palladium-catalyzed C-N cross-coupling with amines, amides, carbamates, and other nitrogen nucleophiles to provide direct access to known and novel rhodamine derivatives, including fluorescent dyes, quenchers, and latent fluorophores.

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