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At least 397 records · Page 22Linked to original sources
PubMed2003

Shedding light on cell signaling: interpretation of FRET biosensors.

This Perspective compares recently developed approaches for studying live-cell signaling dynamics. It is now possible not only to study the changing localizations of proteins within living cells but to correlate these dynamics with quantitation of protein activities, including ligand interactions and phosphorylation. Initial applications of an increasingly varied toolchest of techniques have revealed strengths and weaknesses in each approach, clarifying which tool is best for which job.

Animals↗
PubMed2005

Beta-adrenergic- and muscarinic receptor-induced changes in cAMP activity in adult cardiac myocytes detected with FRET-based biosensor.

beta-Adrenergic receptor activation regulates cardiac myocyte function through the stimulation of cAMP production and subsequent activation of protein kinase A (PKA). Furthermore, muscarinic receptor activation inhibits as well as facilitates these cAMP-dependent effects. However, it has not always been possible to correlate the muscarinic responses with the direct measurement of changes in cellular cAMP activity. Genetically encoded biosensors have recently been developed, making it possible to monitor real-time changes in cAMP and PKA activity at the single cell level. One such biosensor consists of the regulatory and catalytic subunits of PKA labeled with cyan and yellow fluorescent proteins, respectively. Changes in cAMP activity affecting the association of these labeled PKA subunits can be detected as changes in fluorescence resonance energy transfer. In the present study, an adenovirus-based approach was developed to express this recombinant protein complex in adult cardiac myocytes and use it to monitor changes in cAMP activity produced by beta-adrenergic and muscarinic receptor activation. The biosensor expressed with the use of this system is able to detect changes in cAMP activity produced by physiologically relevant levels of beta-adrenergic receptor activation without disrupting normal functional responses. It was also possible to directly demonstrate the complex temporal pattern of inhibitory and stimulatory changes in cAMP activity produced by muscarinic receptor activation in these cells. The adenovirus-based approach we have developed should facilitate the use of this biosensor in studying cAMP and PKA-dependent signaling mechanisms in a wide variety of cell types.

Adenoviridae↗
PubMed2000

Development and application of a GFP-FRET intracellular caspase assay for drug screening.

Apoptosis is a crucial biological process, and activation of caspase endoproteases is essential for proper regulation and execution of apoptosis. Because caspases also appear to be central players in several pathological states, there is a practical need within the biopharmaceutical research community for facile, noninvasive cellular assays for the discovery of compounds that modulate caspase activity. Tandem molecules of green fluorescent protein (GFP) stably expressed within cells can serve as a genetically encoded sensor of protease activity. Using this technology, we have developed a stable cellular system for the screening of agents that modulate activation of the caspase cascade. This assay technology allows for the real-time monitoring of apoptosis in situ, using conventional fluorescent plate reader detection. By applying this assay system to an actual compound screen, small-molecule inducers of cell apoptosis were reliably identified. Follow-up pharmacology confirmed that the rank-order potency of primary hits using the intracellular GFP assay corresponded to that found using a conventional, cell lysis-based assay method.

Amino Acid Sequence↗
PubMed2003

Evaluation of an imaging platform during the development of a FRET protease assay.

Synthetic peptide substrates labeled with a fluorescent donor and quenching moiety flanking an enzyme cleavage site provide a reliable method for monitoring enzyme activity. The dye pair Mca/Dnp has been widely used for this purpose, but poor solubility characteristics, combined with fluorescence emission in the region of the spectrum associated with interference from biologicals and library compounds, can limit the usefulness of Mca/Dnp substrates in a high-throughput screening (HTS) environment. Peptide Mca-Arg-Pro-Lys-Pro-Val-Glu-Nva-Trp-Arg-Lys(Dnp)-NH(2) is a matrix-metalloproteinase 3 (MMP-3) enzyme substrate that the authors have labeled with a CyDye pair, Cy3/Cy5Q. The Mca/Dnp- and CyDye-labeled substrates were compared during the development of an MMP-3 inhibitor assay. The results obtained showed that although the peptide substrates behaved similarly throughout the development of the MMP-3 assay, during a test screen of 934 compounds randomly selected from a collection of more than 70000 compounds, the CyDye substrate was considerably more reliable. Screen Z factor values of 0.84 and 0.15 were obtained using the CyDye and Mca/Dnp peptides respectively, and the authors found that although < 1% of the test compounds were auto-fluorescent at Cy3 wavelengths, > 10% could not be screened using the Mca/Dnp substrate because of compound auto-fluorescence and interference. During this study, the authors used a PMT-based fluorescence plate reader and at the same time evaluated a charged couple device (CCD)-based imaging platform specifically optimized for use with CyDye reagents. The imaging platform gave improved read accuracy and faster plate processing times compared with the PMT reader. Overall, the results presented here highlight the potential benefit of employing the red-shifted CyDye reagents and imaging technology during the development and execution of HTS protease screens.

Fluorescence↗
PubMed2006

Identification of the JAK2 V617F mutation in chronic myeloproliferative disorders using FRET probes and melting curve analysis.

We developed and validated a real-time polymerase chain reaction assay using fluorescent hybridization probes and melting curve analysis to identify the JAK2 V617F mutation, which is implicated in a substantial proportion of chronic myeloproliferative disorders (CMPDs). DNA from 161 samples was isolated from peripheral blood granulocytes and formalin-fixed bone marrow clot sections in patients with CMPDs and without myeloproliferative disorders previously genotyped for the JAK2 V617F (G-->T) mutation, which included 114 wild types (GG) and 47 mutants (GT and TT). Melting curve analysis of these samples yielded 114 wild types, 42 heterozygotes, and 5 homozygotes showing 100% concordance. Analytic sensitivity of the assay for mutant DNA was 5% for the LightTyper (Roche Applied Sciences, Indianapolis, IN) and 10% for the LightCycler (Roche Applied Sciences). Consistent with earlier reports, 78% of the non-chronic myelogenous leukemia CMPD patients and 8% of non-CMPD patients displayed this mutation. This study demonstrates that clinical genotyping of the JAK2 V617F mutation can be performed by melting analysis using both freshly isolated and formalin-fixed tissues.

Bone Marrow Cells↗
PubMed2005

Application of microscope-based FRET to study molecular interactions in focal adhesions of live cells.

This chapter describes the use of microscope-based fluorescence resonance energy transfer to follow dynamic interaction of molecules localized at focal adhesions. We first outline the significance of studying dynamic interactions in focal adhesions of living cells, and second provide an overview of the method itself. This is followed by a protocol for fluorescence resonance energy transfer measurements in live cells.

Animals↗
PubMed2004

Quantification of Protein-Lipid Selectivity using FRET: Application to the M13 Major Coat Protein.

Quantification of lipid selectivity by membrane proteins has been previously addressed mainly from electron spin resonance studies. We present here a new methodology for quantification of protein-lipid selectivity based on fluorescence resonance energy transfer. A mutant of M13 major coat protein was labeled with 7-diethylamino-3((4'iodoacetyl)amino)phenyl-4-methylcoumarin to be used as the donor in energy transfer studies. Phospholipids labeled with N-(7-nitro-2-1,3-benzoxadiazol-4-yl) were selected as the acceptors. The dependence of protein-lipid selectivity on both hydrophobic mismatch and headgroup family was determined. M13 major coat protein exhibited larger selectivity toward phospholipids which allow for a better hydrophobic matching. Increased selectivity was also observed for anionic phospholipids and the relative association constants agreed with the ones already presented in the literature and obtained through electron spin resonance studies. This result led us to conclude that fluorescence resonance energy transfer is a promising methodology in protein-lipid selectivity studies.

Algorithms↗
PubMed2005

Single-molecule FRET studies of important intermediates in the nucleocapsid-protein-chaperoned minus-strand transfer step in HIV-1 reverse transcription.

The minus-strand transfer step of HIV-1 reverse transcription is chaperoned by the nucleocapsid protein (NC), which has been shown to facilitate the annealing between the transactivation response element (TAR) RNA and complementary TAR DNA stem-loop structures. In this work, potential intermediates in the mechanism of NC-chaperoned TAR DNA/TAR RNA annealing have been examined using single-molecule fluorescence resonance energy transfer. The interaction between TAR DNA and various DNA oligonucleotides designed to mimic the initial annealing step was monitored to capture potential intermediates along the reaction pathway. Two possible mechanisms of annealing were examined, namely nucleation through the 3'/5' termini, termed the "zipper" complex, or nucleation through the hairpin loops in a "kissing" complex. Intermediates associated with both mechanisms were observed in the presence of NC, and the kinetics of formation of these intermediates were also measured. Thus, the single-molecule experiments support the notion that NC-assisted annealing of TAR DNA:TAR RNA may occur through multiple pathways.

Base Sequence↗
PubMed2005

Conformational changes involved in MscL channel gating measured using FRET spectroscopy.

We demonstrate that fluorescence resonance energy transfer spectroscopy is a powerful tool for in situ structural analysis of multimeric membrane proteins by measuring the conformational changes involved in gating the mechanosensitive ion channel of large conductance. Ensemble analysis is used to analyze the intensity of light emitted by AlexaFluor-labeled cysteine mutants reconstituted into artificial liposomes before and after acceptor photobleaching. The diameter of the protein is found to increase by 16 A upon channel activation.

Escherichia coli Proteins↗
PubMed2006

Conformational heterogeneity in RNA polymerase observed by single-pair FRET microscopy.

Kinetic, structural, and single-molecule transcription measurements suggest that RNA polymerase can adopt many different conformations during elongation. We have measured the geometry of the DNA and RNA in ternary elongation complexes using single-pair fluorescence resonance energy transfer. Six different synthetic transcription elongation complexes were constructed from DNA containing an artificial transcription bubble, an RNA primer, and core RNA polymerase from Escherichia coli. Two different RNA primers were used, an 8-mer and a 5'-extended 11-mer. Fluorescent dye labels were attached at one of three positions on the DNA and at the RNA primer 5'-end. Structurally, the upstream DNA runs perpendicular to the proposed RNA exit channel. Upon nucleoside-triphosphate addition, DNA/RNA hybrid separation occurs readily in the 11-mer complexes but not in the 8-mer complexes. Clear evidence was obtained that RNA polymerase exists in multiple conformations among which it fluctuates.

Computer Simulation↗