The Experts below are selected from a list of 188415 Experts worldwide ranked by ideXlab platform

Roger Y Tsien - One of the best experts on this subject based on the ideXlab platform.

  • a far red Fluorescent Protein evolved from a cyanobacterial phycobiliProtein
    Nature Methods, 2016
    Co-Authors: Erik A Rodriguez, Roger Y Tsien, Geraldine N Tran, Larry A Gross, Jessica L Crisp, Xiaokun Shu, John Y Lin
    Abstract:

    A bright and photostable far-red Fluorescent Protein, smURFP, was developed from a cyanobacterial phycobiliProtein. smURFP uniquely binds a highly cell-permeable biliverdin derivative to obtain fluorescence brightness comparable to that of eGFP in cells.

  • improved monomeric red orange and yellow Fluorescent Proteins derived from discosoma sp red Fluorescent Protein
    Nature Biotechnology, 2004
    Co-Authors: Nathan C. Shaner, Robert E. Campbell, Paul Steinbach, Ben N G Giepmans, Amy E Palmer, Roger Y Tsien
    Abstract:

    Improved monomeric red, orange and yellow Fluorescent Proteins derived from Discosoma sp. red Fluorescent Protein

  • a monomeric red Fluorescent Protein
    Proceedings of the National Academy of Sciences of the United States of America, 2002
    Co-Authors: Robert E. Campbell, Paul Steinbach, Amy E Palmer, Oded Tour, Geoffrey S Baird, David A Zacharias, Roger Y Tsien
    Abstract:

    All coelenterate Fluorescent Proteins cloned to date display some form of quaternary structure, including the weak tendency of Aequorea green Fluorescent Protein (GFP) to dimerize, the obligate dimerization of Renilla GFP, and the obligate tetramerization of the red Fluorescent Protein from Discosoma (DsRed). Although the weak dimerization of Aequorea GFP has not impeded its acceptance as an indispensable tool of cell biology, the obligate tetramerization of DsRed has greatly hindered its use as a genetically encoded fusion tag. We present here the stepwise evolution of DsRed to a dimer and then either to a genetic fusion of two copies of the Protein, i.e., a tandem dimer, or to a true monomer designated mRFP1 (monomeric red Fluorescent Protein). Each subunit interface was disrupted by insertion of arginines, which initially crippled the resulting Protein, but red fluorescence could be rescued by random and directed mutagenesis totaling 17 substitutions in the dimer and 33 in mRFP1. Fusions of the gap junction Protein connexin43 to mRFP1 formed fully functional junctions, whereas analogous fusions to the tetramer and dimer failed. Although mRFP1 has somewhat lower extinction coefficient, quantum yield, and photostability than DsRed, mRFP1 matures >10 times faster, so that it shows similar brightness in living cells. In addition, the excitation and emission peaks of mRFP1, 584 and 607 nm, are ≈25 nm red-shifted from DsRed, which should confer greater tissue penetration and spectral separation from autofluorescence and other Fluorescent Proteins.

  • reducing the environmental sensitivity of yellow Fluorescent Protein mechanism and applications
    Journal of Biological Chemistry, 2001
    Co-Authors: Oliver Griesbeck, Robert E. Campbell, Geoffrey S Baird, David A Zacharias, Roger Y Tsien
    Abstract:

    Yellow mutants of the green Fluorescent Protein (YFP) are crucial constituents of genetically encoded indicators of signal transduction and fusions to monitor ProteinProtein interactions. However, previous YFPs show excessive pH sensitivity, chloride interference, poor photostability, or poor expression at 37 °C. Protein evolution in Escherichia coli has produced a new YFP named Citrine, in which the mutation Q69M confers a much lower pKa (5.7) than for previous YFPs, indifference to chloride, twice the photostability of previous YFPs, and much better expression at 37 °C and in organelles. The halide resistance is explained by a 2.2-A x-ray crystal structure of Citrine, showing that the methionine side chain fills what was once a large halide-binding cavity adjacent to the chromophore. Insertion of calmodulin within Citrine or fusion of cyan Fluorescent Protein, calmodulin, a calmodulin-binding peptide and Citrine has generated improved calcium indicators. These chimeras can be targeted to multiple cellular locations and have permitted the first single-cell imaging of free [Ca 21 ] in the Golgi. Citrine is superior to all previous YFPs except when pH or halide sensitivity is desired and is particularly advantageous within genetically encoded Fluorescent indicators of physiological signals.

  • biochemistry mutagenesis and oligomerization of dsred a red Fluorescent Protein from coral
    Proceedings of the National Academy of Sciences of the United States of America, 2000
    Co-Authors: Geoffrey S Baird, David A Zacharias, Roger Y Tsien
    Abstract:

    Abstract DsRed is a recently cloned 28-kDa Fluorescent Protein responsible for the red coloration around the oral disk of a coral of the Discosoma genus. DsRed has attracted tremendous interest as a potential expression tracer and fusion partner that would be complementary to the homologous green Fluorescent Protein from Aequorea, but very little is known of the biochemistry of DsRed. We now show that DsRed has a much higher extinction coefficient and quantum yield than previously reported, plus excellent resistance to pH extremes and photobleaching. In addition, its 583-nm emission maximum can be further shifted to 602 nm by mutation of Lys-83 to Met. However, DsRed has major drawbacks, such as strong oligomerization and slow maturation. Analytical ultracentrifugation proves DsRed to be an obligate tetramer in vitro, and fluorescence resonance energy transfer measurements and yeast two-hybrid assays verify oligomerization in live cells. Also, DsRed takes days to ripen fully from green to red in vitro or in vivo, and mutations such as Lys-83 to Arg prevent the color change. Many potential cell biological applications of DsRed will require suppression of the tetramerization and acceleration of the maturation.

Theodorus W J Gadella - One of the best experts on this subject based on the ideXlab platform.

  • structure of a Fluorescent Protein from aequorea victoria bearing the obligate monomer mutation a206k
    Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2012
    Co-Authors: D Von Stetten, Joachim Goedhart, Theodorus W J Gadella, Marjolaine Noirclercsavoye, Antoine Royant
    Abstract:

    The green Fluorescent Protein (GFP) from the jellyfish Aequoria victoria has been shown to dimerize at high concentrations, which could lead to artefacts in imaging experiments. To ensure a truly monomeric state, an A206K mutation has been introduced into most of its widely used variants, with minimal effect on the spectroscopic properties. Here, the first structure of one of these variants, the cyan Fluorescent Protein mTurquoise, is presented and compared with that of its dimeric version mTurquoise-K206A. No significant structural change is detected in the chromophore cavity, reinforcing the notion that this mutation is spectroscopically silent and validating that the structural analysis performed on dimeric mutants also applies to monomeric versions. Finally, it is explained why cyan versions of GFP containing the Y66W and N146I mutations do not require the A206K mutation to prevent dimerization at high concentrations.

  • bright cyan Fluorescent Protein variants identified by fluorescence lifetime screening
    Nature Methods, 2010
    Co-Authors: Joachim Goedhart, Mark A. Hink, Laura Van Weeren, Norbert O E Vischer, Kees Jalink, Theodorus W J Gadella
    Abstract:

    Lifetime screening of Fluorescent Protein variants by Fluorescent lifetime imaging microscopy of bacterial colonies identifies bright, high-quantum-yield Fluorescent Protein variants including a cyan Fluorescent Protein named mTurquoise that is 1.5-fold brighter than mCerulean and has a mono-exponential fluorescence decay.

  • sensitive detection of p65 homodimers using red shifted and Fluorescent Protein based fret couples
    PLOS ONE, 2007
    Co-Authors: Joachim Goedhart, Laura Van Weeren, Joop E M Vermeer, Merel J W Adjobohermans, Theodorus W J Gadella
    Abstract:

    BackgroundFluorescence Resonance Energy Transfer (FRET) between the green Fluorescent Protein (GFP) variants CFP and YFP is widely used for the detection of Protein-Protein interactions. Nowadays, several monomeric red-shifted Fluorescent Proteins are available that potentially improve the efficiency of FRET.Methodology/Principal FindingsTo allow side-by-side comparison of several Fluorescent Protein combinations for detection of FRET, yellow or orange Fluorescent Proteins were directly fused to red Fluorescent Proteins. FRET from yellow Fluorescent Proteins to red Fluorescent Proteins was detected by both FLIM and donor dequenching upon acceptor photobleaching, showing that mCherry and mStrawberry were more efficient acceptors than mRFP1. Circular permutated yellow Fluorescent Protein variants revealed that in the tandem constructs the orientation of the transition dipole moment influences the FRET efficiency. In addition, it was demonstrated that the orange Fluorescent Proteins mKO and mOrange are both suitable as donor for FRET studies. The most favorable orange-red FRET pair was mKO-mCherry, which was used to detect homodimerization of the NF-κB subunit p65 in single living cells, with a threefold higher lifetime contrast and a twofold higher FRET efficiency than for CFP-YFP.Conclusions/SignificanceThe observed high FRET efficiency of red-shifted couples is in accordance with increased Forster radii of up to 64 A, being significantly higher than the Forster radius of the commonly used CFP-YFP pair. Thus, red-shifted FRET pairs are preferable for detecting Protein-Protein interactions by donor-based FRET methods in single living cells.

  • bright monomeric red Fluorescent Protein with an extended fluorescence lifetime
    Nature Methods, 2007
    Co-Authors: Ekaterina M. Merzlyak, Sergey Lukyanov, Dmitry Shcherbo, Arkady F. Fradkov, Konstantin A. Lukyanov, Joachim Goedhart, Mariya E Bulina, Aleksandr S Shcheglov, Anna Gaintzeva, Theodorus W J Gadella
    Abstract:

    Fluorescent Proteins have become extremely popular tools for in vivo imaging and especially for the study of localization, motility and interaction of Proteins in living cells. Here we report TagRFP, a monomeric red Fluorescent Protein, which is characterized by high brightness, complete chromophore maturation, prolonged fluorescence lifetime and high pH-stability. These properties make TagRFP an excellent tag for Protein localization studies and fluorescence resonance energy transfer (FRET) applications.

  • bright monomeric red Fluorescent Protein with an extended fluorescence lifetime
    Nature Methods, 2007
    Co-Authors: Ekaterina M. Merzlyak, Sergey Lukyanov, Dmitry Shcherbo, Arkady F. Fradkov, Konstantin A. Lukyanov, Joachim Goedhart, Mariya E Bulina, Aleksandr S Shcheglov, Anna Gaintzeva, Theodorus W J Gadella
    Abstract:

    Fluorescent Proteins have become extremely popular tools for in vivo imaging and especially for the study of localization, motility and interaction of Proteins in living cells. Here we report TagRFP, a monomeric red Fluorescent Protein, which is characterized by high brightness, complete chromophore maturation, prolonged fluorescence lifetime and high pH-stability. These properties make TagRFP an excellent tag for Protein localization studies and fluorescence resonance energy transfer (FRET) applications.

Joachim Goedhart - One of the best experts on this subject based on the ideXlab platform.

  • structure of a Fluorescent Protein from aequorea victoria bearing the obligate monomer mutation a206k
    Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2012
    Co-Authors: D Von Stetten, Joachim Goedhart, Theodorus W J Gadella, Marjolaine Noirclercsavoye, Antoine Royant
    Abstract:

    The green Fluorescent Protein (GFP) from the jellyfish Aequoria victoria has been shown to dimerize at high concentrations, which could lead to artefacts in imaging experiments. To ensure a truly monomeric state, an A206K mutation has been introduced into most of its widely used variants, with minimal effect on the spectroscopic properties. Here, the first structure of one of these variants, the cyan Fluorescent Protein mTurquoise, is presented and compared with that of its dimeric version mTurquoise-K206A. No significant structural change is detected in the chromophore cavity, reinforcing the notion that this mutation is spectroscopically silent and validating that the structural analysis performed on dimeric mutants also applies to monomeric versions. Finally, it is explained why cyan versions of GFP containing the Y66W and N146I mutations do not require the A206K mutation to prevent dimerization at high concentrations.

  • bright cyan Fluorescent Protein variants identified by fluorescence lifetime screening
    Nature Methods, 2010
    Co-Authors: Joachim Goedhart, Mark A. Hink, Laura Van Weeren, Norbert O E Vischer, Kees Jalink, Theodorus W J Gadella
    Abstract:

    Lifetime screening of Fluorescent Protein variants by Fluorescent lifetime imaging microscopy of bacterial colonies identifies bright, high-quantum-yield Fluorescent Protein variants including a cyan Fluorescent Protein named mTurquoise that is 1.5-fold brighter than mCerulean and has a mono-exponential fluorescence decay.

  • sensitive detection of p65 homodimers using red shifted and Fluorescent Protein based fret couples
    PLOS ONE, 2007
    Co-Authors: Joachim Goedhart, Laura Van Weeren, Joop E M Vermeer, Merel J W Adjobohermans, Theodorus W J Gadella
    Abstract:

    BackgroundFluorescence Resonance Energy Transfer (FRET) between the green Fluorescent Protein (GFP) variants CFP and YFP is widely used for the detection of Protein-Protein interactions. Nowadays, several monomeric red-shifted Fluorescent Proteins are available that potentially improve the efficiency of FRET.Methodology/Principal FindingsTo allow side-by-side comparison of several Fluorescent Protein combinations for detection of FRET, yellow or orange Fluorescent Proteins were directly fused to red Fluorescent Proteins. FRET from yellow Fluorescent Proteins to red Fluorescent Proteins was detected by both FLIM and donor dequenching upon acceptor photobleaching, showing that mCherry and mStrawberry were more efficient acceptors than mRFP1. Circular permutated yellow Fluorescent Protein variants revealed that in the tandem constructs the orientation of the transition dipole moment influences the FRET efficiency. In addition, it was demonstrated that the orange Fluorescent Proteins mKO and mOrange are both suitable as donor for FRET studies. The most favorable orange-red FRET pair was mKO-mCherry, which was used to detect homodimerization of the NF-κB subunit p65 in single living cells, with a threefold higher lifetime contrast and a twofold higher FRET efficiency than for CFP-YFP.Conclusions/SignificanceThe observed high FRET efficiency of red-shifted couples is in accordance with increased Forster radii of up to 64 A, being significantly higher than the Forster radius of the commonly used CFP-YFP pair. Thus, red-shifted FRET pairs are preferable for detecting Protein-Protein interactions by donor-based FRET methods in single living cells.

  • bright monomeric red Fluorescent Protein with an extended fluorescence lifetime
    Nature Methods, 2007
    Co-Authors: Ekaterina M. Merzlyak, Sergey Lukyanov, Dmitry Shcherbo, Arkady F. Fradkov, Konstantin A. Lukyanov, Joachim Goedhart, Mariya E Bulina, Aleksandr S Shcheglov, Anna Gaintzeva, Theodorus W J Gadella
    Abstract:

    Fluorescent Proteins have become extremely popular tools for in vivo imaging and especially for the study of localization, motility and interaction of Proteins in living cells. Here we report TagRFP, a monomeric red Fluorescent Protein, which is characterized by high brightness, complete chromophore maturation, prolonged fluorescence lifetime and high pH-stability. These properties make TagRFP an excellent tag for Protein localization studies and fluorescence resonance energy transfer (FRET) applications.

  • bright monomeric red Fluorescent Protein with an extended fluorescence lifetime
    Nature Methods, 2007
    Co-Authors: Ekaterina M. Merzlyak, Sergey Lukyanov, Dmitry Shcherbo, Arkady F. Fradkov, Konstantin A. Lukyanov, Joachim Goedhart, Mariya E Bulina, Aleksandr S Shcheglov, Anna Gaintzeva, Theodorus W J Gadella
    Abstract:

    Fluorescent Proteins have become extremely popular tools for in vivo imaging and especially for the study of localization, motility and interaction of Proteins in living cells. Here we report TagRFP, a monomeric red Fluorescent Protein, which is characterized by high brightness, complete chromophore maturation, prolonged fluorescence lifetime and high pH-stability. These properties make TagRFP an excellent tag for Protein localization studies and fluorescence resonance energy transfer (FRET) applications.

Robert E. Campbell - One of the best experts on this subject based on the ideXlab platform.

  • phuji a ph sensitive red Fluorescent Protein for imaging of exo and endocytosis
    Journal of Cell Biology, 2014
    Co-Authors: Yi Shen, Robert E. Campbell, Morgane Rosendale, David Perrais
    Abstract:

    Fluorescent Proteins with pH-sensitive fluorescence are valuable tools for the imaging of exocytosis and endocytosis. The Aequorea green Fluorescent Protein mutant superecliptic pHluorin (SEP) is particularly well suited to these applications. Here we describe pHuji, a red Fluorescent Protein with a pH sensitivity that approaches that of SEP, making it amenable for detection of single exocytosis and endocytosis events. To demonstrate the utility of the pHuji plus SEP pair, we perform simultaneous two-color imaging of clathrin-mediated internalization of both the transferrin receptor and the β2 adrenergic receptor. These experiments reveal that the two receptors are differentially sorted at the time of endocytic vesicle formation.

  • a fluorogenic red Fluorescent Protein heterodimer
    Chemistry & Biology, 2012
    Co-Authors: Spencer C Alford, Ahmed S Abdelfattah, Yidan Ding, Robert E. Campbell
    Abstract:

    Summary The expanding repertoire of genetically encoded biosensors constructed from variants of Aequorea victoria green Fluorescent Protein (GFP) enable the imaging of a variety of intracellular biochemical processes. To facilitate the imaging of multiple biosensors in a single cell, we undertook the development of a dimerization-dependent red Fluorescent Protein (ddRFP) that provides an alternative strategy for biosensor construction. An extensive process of rational engineering and directed Protein evolution led to the discovery of a ddRFP with a K d of 33 μM and a 10-fold increase in fluorescence upon heterodimer formation. We demonstrate that the dimerization-dependent fluorescence of ddRFP can be used for detection of a Protein-Protein interaction in vitro, imaging of the reversible Ca 2+ -dependent association of calmodulin and M13 in live cells, and imaging of caspase-3 activity during apoptosis.

  • improved monomeric red orange and yellow Fluorescent Proteins derived from discosoma sp red Fluorescent Protein
    Nature Biotechnology, 2004
    Co-Authors: Nathan C. Shaner, Robert E. Campbell, Paul Steinbach, Ben N G Giepmans, Amy E Palmer, Roger Y Tsien
    Abstract:

    Improved monomeric red, orange and yellow Fluorescent Proteins derived from Discosoma sp. red Fluorescent Protein

  • a monomeric red Fluorescent Protein
    Proceedings of the National Academy of Sciences of the United States of America, 2002
    Co-Authors: Robert E. Campbell, Paul Steinbach, Amy E Palmer, Oded Tour, Geoffrey S Baird, David A Zacharias, Roger Y Tsien
    Abstract:

    All coelenterate Fluorescent Proteins cloned to date display some form of quaternary structure, including the weak tendency of Aequorea green Fluorescent Protein (GFP) to dimerize, the obligate dimerization of Renilla GFP, and the obligate tetramerization of the red Fluorescent Protein from Discosoma (DsRed). Although the weak dimerization of Aequorea GFP has not impeded its acceptance as an indispensable tool of cell biology, the obligate tetramerization of DsRed has greatly hindered its use as a genetically encoded fusion tag. We present here the stepwise evolution of DsRed to a dimer and then either to a genetic fusion of two copies of the Protein, i.e., a tandem dimer, or to a true monomer designated mRFP1 (monomeric red Fluorescent Protein). Each subunit interface was disrupted by insertion of arginines, which initially crippled the resulting Protein, but red fluorescence could be rescued by random and directed mutagenesis totaling 17 substitutions in the dimer and 33 in mRFP1. Fusions of the gap junction Protein connexin43 to mRFP1 formed fully functional junctions, whereas analogous fusions to the tetramer and dimer failed. Although mRFP1 has somewhat lower extinction coefficient, quantum yield, and photostability than DsRed, mRFP1 matures >10 times faster, so that it shows similar brightness in living cells. In addition, the excitation and emission peaks of mRFP1, 584 and 607 nm, are ≈25 nm red-shifted from DsRed, which should confer greater tissue penetration and spectral separation from autofluorescence and other Fluorescent Proteins.

  • reducing the environmental sensitivity of yellow Fluorescent Protein mechanism and applications
    Journal of Biological Chemistry, 2001
    Co-Authors: Oliver Griesbeck, Robert E. Campbell, Geoffrey S Baird, David A Zacharias, Roger Y Tsien
    Abstract:

    Yellow mutants of the green Fluorescent Protein (YFP) are crucial constituents of genetically encoded indicators of signal transduction and fusions to monitor ProteinProtein interactions. However, previous YFPs show excessive pH sensitivity, chloride interference, poor photostability, or poor expression at 37 °C. Protein evolution in Escherichia coli has produced a new YFP named Citrine, in which the mutation Q69M confers a much lower pKa (5.7) than for previous YFPs, indifference to chloride, twice the photostability of previous YFPs, and much better expression at 37 °C and in organelles. The halide resistance is explained by a 2.2-A x-ray crystal structure of Citrine, showing that the methionine side chain fills what was once a large halide-binding cavity adjacent to the chromophore. Insertion of calmodulin within Citrine or fusion of cyan Fluorescent Protein, calmodulin, a calmodulin-binding peptide and Citrine has generated improved calcium indicators. These chimeras can be targeted to multiple cellular locations and have permitted the first single-cell imaging of free [Ca 21 ] in the Golgi. Citrine is superior to all previous YFPs except when pH or halide sensitivity is desired and is particularly advantageous within genetically encoded Fluorescent indicators of physiological signals.

David W Piston - One of the best experts on this subject based on the ideXlab platform.

  • high contrast imaging of Fluorescent Protein fret by fluorescence polarization microscopy
    Biophysical Journal, 2005
    Co-Authors: Mark A. Rizzo, David W Piston
    Abstract:

    Detection of Forster resonance energy transfer (FRET) between Fluorescent Protein labeled targets is a valuable strategy for measurement of Protein-Protein interactions and other intracellular processes. Despite the utility of FRET, widespread application of this technique to biological problems and high-throughput screening has been limited by low-contrast measurement strategies that rely on the detection of sensitized emission or photodestruction of the sample. Here we report a FRET detection strategy based on detecting depolarized sensitized emission. In the absence of FRET, we show that fluorescence emission from a donor Fluorescent Protein is highly polarized. Depolarization of fluorescence emission is observed only in the presence of energy transfer. A simple detection strategy was adapted for fluorescence microscopy using both laser scanning and wide-field approaches. This approach is able to distinguish FRET between linked and unlinked Cerulean and Venus Fluorescent Proteins in living cells with a larger dynamic range than other approaches.

  • an improved cyan Fluorescent Protein variant useful for fret
    Nature Biotechnology, 2004
    Co-Authors: Mark A. Rizzo, Gerald Springer, Butch Granada, David W Piston
    Abstract:

    Many genetically encoded biosensors use Forster resonance energy transfer (FRET) between Fluorescent Proteins to report biochemical phenomena in living cells. Most commonly, the enhanced cyan Fluorescent Protein (ECFP) is used as the donor fluorophore, coupled with one of several yellow Fluorescent Protein (YFP) variants as the acceptor. ECFP is used despite several spectroscopic disadvantages, namely a low quantum yield, a low extinction coefficient and a fluorescence lifetime that is best fit by a double exponential. To improve the characteristics of ECFP for FRET measurements, we used a site-directed mutagenesis approach to overcome these disadvantages. The resulting variant, which we named Cerulean (ECFP/S72A/Y145A/H148D), has a greatly improved quantum yield, a higher extinction coefficient and a fluorescence lifetime that is best fit by a single exponential. Cerulean is 2.5-fold brighter than ECFP and replacement of ECFP with Cerulean substantially improves the signal-to-noise ratio of a FRET-based sensor for glucokinase activation.

  • Fluorescent Protein spectra
    Journal of Cell Science, 2001
    Co-Authors: G. H. Patterson, Rich N. Day, David W Piston
    Abstract:

    The cloning of the green Fluorescent Protein (GFP) from the jellyfish Aequoria victoria and its expression in heterologous systems was a significant advance for optical microscopy of living cells ([Chalfie et al., 1994][1]). Mutagenesis of jellyfish GFP has yielded Proteins that fluoresce from blue