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131 Publications

Showing 111-120 of 131 results
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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12/01/11 | Advances in the chemistry of small molecule fluorescent probes.
Wysocki LM, Lavis LD
Current Opinion in Chemical Biology. 2011 Dec;15(6):752-9. doi: 10.1016/j.cbpa.2011.10.013

Small molecule fluorophores are essential tools for chemical biology. A benefit of synthetic dyes is the ability to employ chemical approaches to control the properties and direct the position of the fluorophore. Applying modern synthetic organic chemistry strategies enables efficient tailoring of the chemical structure to obtain probes for specific biological experiments. Chemistry can also be used to activate fluorophores; new fluorogenic enzyme substrates and photoactivatable compounds with improved properties have been prepared that facilitate advanced imaging experiments with low background fluorescence. Finally, chemical reactions in live cells can be used to direct the spatial distribution of the fluorophore, allowing labeling of defined cellular regions with synthetic dyes.

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11/18/11 | Facile and general synthesis of photoactivatable xanthene dyes.
Wysocki LM, Grimm JB, Tkachuk AN, Brown TA, Betzig E, Lavis LD
Angewandte Chemie. 2011 Nov 18;50:11206-9. doi: 10.1002/anie.201104571

Despite the apparent simplicity of the xanthene fluorophores, the preparation of caged derivatives with free carboxy groups remains a synthetic challenge. A straightforward and flexible strategy for preparing rhodamine and fluorescein derivatives was developed using reduced, “leuco” intermediates.

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02/01/11 | Histochemistry: live and in color.
Lavis LD
The Journal of Histochemistry and Cytochemistry: Official Journal of the Histochemistry Society. 2011 Feb;59:139-45. doi: 10.1369/0022155410395760

Histochemistry (chemistry in the context of biological tissue) is an invaluable set of techniques used to visualize biological structures. This field lies at the interface of organic chemistry, biochemistry, and biology. Integration of these disciplines over the past century has permitted the imaging of cells and tissues using microscopy. Today, by exploiting the unique chemical environments within cells, heterologous expression techniques, and enzymatic activity, histochemical methods can be used to visualize structures in living matter. This review focuses on the labeling techniques and organic fluorophores used in live cells.

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01/01/11 | Synthesis and utility of fluorogenic acetoxymethyl ethers.
Lavis LD, Chao T, Raines RT
Chemical Science. 2011 Jan 1;2(3):521-30. doi: 10.1039/C0SC00466A

Phenolic fluorophores such as fluorescein, Tokyo Green, resorufin, and their derivatives are workhorses of biological science. Acylating the phenolic hydroxyl group(s) in these fluorophores masks their fluorescence. The ensuing ester is a substrate for cellular esterases, which can restore fluorescence. These esters are, however, notoriously unstable to hydrolysis, severely compromising their utility. The acetoxymethyl (AM) group is an esterase-sensitive motif that can mask polar functionalities in small molecules. Here, we report on the use of AM ether groups to mask phenolic fluorophores. The resulting profluorophores have a desirable combination of low background fluorescence, high chemical stability, and high enzymatic reactivity, both in vitro and in cellulo. These simple phenyl ether-based profluorophores could supplement or supplant the use of phenyl esters for imaging biochemical and biological systems.

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12/21/10 | Cellular uptake of ribonuclease A relies on anionic glycans.
Chao T, Lavis LD, Raines RT
Biochemistry. 2010 Dec 21;49(50):10666-73. doi: 10.1021/bi1013485

Bovine pancreatic ribonuclease (RNase A) can enter human cells, even though it lacks a cognate cell-surface receptor protein. Here, we report on the biochemical basis for its cellular uptake. Analyses in vitro and in cellulo revealed that RNase A interacts tightly with abundant cell-surface proteoglycans containing glycosaminoglycans, such as heparan sulfate and chondroitin sulfate, as well as with sialic acid-containing glycoproteins. The uptake of RNase A correlates with cell anionicity, as quantified by measuring electrophoretic mobility. The cellular binding and uptake of RNase A contrast with those of Onconase, an amphibian homologue that does not interact tightly with anionic cell-surface glycans. As anionic glycans are especially abundant on human tumor cells, our data predicate utility for mammalian ribonucleases as cancer chemotherapeutic agents.

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