The Experts below are selected from a list of 84837 Experts worldwide ranked by ideXlab platform
Shen Zhang - One of the best experts on this subject based on the ideXlab platform.
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a general strategy to red shift Green Fluorescent Protein based biosensors
Nature Chemical Biology, 2020Co-Authors: Shen ZhangAbstract:Compared with Green Fluorescent Protein-based biosensors, red Fluorescent Protein (RFP)-based biosensors are inherently advantageous because of reduced phototoxicity, decreased autofluorescence and enhanced tissue penetration. However, existing RFP-based biosensors often suffer from small dynamic ranges, mislocalization and undesired photoconversion. In addition, the choice of available RFP-based biosensors is limited, and development of each biosensor requires substantial effort. Herein, we describe a general and convenient method, which introduces a genetically encoded noncanonical amino acid, 3-aminotyrosine, to the chromophores of Green Fluorescent Protein-like Proteins and biosensors for spontaneous and efficient Green-to-red conversion. We demonstrated that this method could be used to quickly expand the repertoire of RFP-based biosensors. With little optimization, the 3-aminotyrosine-modified biosensors preserved the molecular brightness, dynamic range and responsiveness of their Green Fluorescent predecessors. We further applied spectrally resolved biosensors for multiplexed imaging of metabolic dynamics in pancreatic β-cells.
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a general strategy to red shift Green Fluorescent Protein based biosensors
bioRxiv, 2020Co-Authors: Shen ZhangAbstract:Compared to Green Fluorescent Protein (GFP) based biosensors, red Fluorescent Protein (RFP) based biosensors are inherently advantageous because of reduced phototoxicity, decreased autofluorescence, and enhanced tissue penetration. However, there is a limited choice of RFP-based biosensors and development of each biosensor requires significant effort. Herein, we describe a general and convenient method which uses the genetically encoded amino acid, 3-aminotyrosine (aY), to convert GFPs and GFP-based biosensors into red.
Steven G Boxer - One of the best experts on this subject based on the ideXlab platform.
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mechanism of color and photoacidity tuning for the protonated Green Fluorescent Protein chromophore
Journal of the American Chemical Society, 2020Co-Authors: Steven G BoxerAbstract:The neutral or A state of the Green Fluorescent Protein (GFP) chromophore is a remarkable example of a photoacid naturally embedded in the Protein environment and accounts for the large Stokes shif...
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short hydrogen bonds and proton delocalization in Green Fluorescent Protein gfp
ACS central science, 2015Co-Authors: Luke M Oltrogge, Steven G BoxerAbstract:Short hydrogen bonds and specifically low-barrier hydrogen bonds (LBHBs) have been the focus of much attention and controversy for their possible role in enzymatic catalysis. The Green Fluorescent Protein (GFP) mutant S65T, H148D has been found to form a very short hydrogen bond between Asp148 and the chromophore resulting in significant spectral perturbations. Leveraging the unique autocatalytically formed chromophore and its sensitivity to this interaction we explore the consequences of proton affinity matching across this putative LBHB. Through the use of noncanonical amino acids introduced through nonsense suppression or global incorporation, we systematically modify the acidity of the GFP chromophore with halogen substituents. X-ray crystal structures indicated that the length of the interaction with Asp148 is unchanged at ∼2.45 A while the absorbance spectra demonstrate an unprecedented degree of color tuning with increasing acidity. We utilized spectral isotope effects, isotope fractionation factor...
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dynamic stokes shift in Green Fluorescent Protein variants
Proceedings of the National Academy of Sciences of the United States of America, 2007Co-Authors: Paul Abbyad, William Childs, Xinghua Shi, Steven G BoxerAbstract:Solvent reorganization around the excited state of a chromophore leads to an emission shift to longer wavelengths during the excited-state lifetime. This solvation response is absent in wild-type Green Fluorescent Protein, and this has been attributed to rigidity in the chromophore's environment necessary to exclude nonradiative transitions to the ground state. The Fluorescent Protein mPlum was developed via directed evolution by selection for red emission, and we use time-resolved fluorescence to study the dynamic Stokes shift through its evolutionary history. The far-red emission of mPlum is attributed to a picosecond solvation response that is observed at all temperatures above the glass transition. This time-dependent shift in emission is not observed in its evolutionary ancestors, suggesting that selective pressure has produced a chromophore environment that allows solvent reorganization. The evolutionary pathway and structures of related Fluorescent Proteins suggest the role of a single residue in close proximity to the chromophore as the primary cause of the solvation response.
Frederick I. Tsuji - One of the best experts on this subject based on the ideXlab platform.
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A bacterial cloning vector using a mutated Aequorea Green Fluorescent Protein as an indicator
Gene, 1997Co-Authors: Hidesato Ogawa, Kunio Yasuda, Kazuhiko Umesono, Frederick I. TsujiAbstract:The bacterial cloning vector, pGreenscript A, derived from the mutated Aequorea Green Fluorescent Protein (GFP-S65A) gene, when expressed in E. coli produced colonies that showed yellow color under daylight and strong Green fluorescence under longwave ultraviolet light. The vector was used to select for inserted foreign genes based on the loss of the yellow color/Green fluorescence of E. coli cells caused by the insertional inactivation of GFP production.
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aequorea Green Fluorescent Protein expression of the gene and fluorescence characteristics of the recombinant Protein
FEBS Letters, 1994Co-Authors: Frederick I. TsujiAbstract:Expression of the cDNA for Aequorea Green Fluorescent Protein in E. coli yielded a fused Protein with fluorescence excitation and emission spectra virtually identical to those of the native Green Fluorescent Protein. Further, a solution of the Protein, when mixed with aequorin and calcium ion, emitted a Greenish luminescence characteristic of the in vivo luminescence of the animal, indicating a radiationless energy transfer to the Protein.
S J Remington - One of the best experts on this subject based on the ideXlab platform.
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structural and spectral response of Green Fluorescent Protein variants to changes in ph
Biochemistry, 1999Co-Authors: M A Elsliger, Rebekka M. Wachter, George T Hanson, Karen Kallio, S J RemingtonAbstract:The Green Fluorescent Protein (GFP) from the jellyfish Aequorea victoria has become a useful tool in molecular and cell biology. Recently, it has been found that the fluorescence spectra of most mu...
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crystal structure of the aequorea victoria Green Fluorescent Protein
Science, 1996Co-Authors: Mats F Ormo, Roger Y Tsien, Karen Kallio, Andrew B. Cubitt, Larry A Gross, S J RemingtonAbstract:The Green Fluorescent Protein (GFP) from the Pacific Northwest jellyfish Aequorea victoria has generated intense interest as a marker for gene expression and localization of gene products. The chromophore, resulting from the spontaneous cyclization and oxidation of the sequence -Ser65 (or Thr65)-Tyr66-Gly67-, requires the native Protein fold for both formation and fluorescence emission. The structure of Thr65 GFP has been determined at 1.9 angstrom resolution. The Protein fold consists of an 11-stranded beta barrel with a coaxial helix, with the chromophore forming from the central helix. Directed mutagenesis of one residue adjacent to the chromophore, Thr203, to Tyr or His results in significantly red-shifted excitation and emission maxima.
Roger Y Tsien - One of the best experts on this subject based on the ideXlab platform.
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THE Green Fluorescent Protein
Annual Review of Biochemistry, 1998Co-Authors: Roger Y TsienAbstract:In just three years, the Green Fluorescent Protein (GFP) from the jellyfish Aequorea victoria has vaulted from obscurity to become one of the most widely studied and exploited Proteins in biochemistry and cell biology. Its amazing ability to generate a highly visible, efficiently emitting internal fluorophore is both intrinsically fascinating and tremendously valuable. High-resolution crystal structures of GFP offer unprecedented opportunities to understand and manipulate the relation between Protein structure and spectroscopic function. GFP has become well established as a marker of gene expression and Protein targeting in intact cells and organisms. Mutagenesis and engineering of GFP into chimeric Proteins are opening new vistas in physiological indicators, biosensors, and photochemical memories.
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crystal structure of the aequorea victoria Green Fluorescent Protein
Science, 1996Co-Authors: Mats F Ormo, Roger Y Tsien, Karen Kallio, Andrew B. Cubitt, Larry A Gross, S J RemingtonAbstract:The Green Fluorescent Protein (GFP) from the Pacific Northwest jellyfish Aequorea victoria has generated intense interest as a marker for gene expression and localization of gene products. The chromophore, resulting from the spontaneous cyclization and oxidation of the sequence -Ser65 (or Thr65)-Tyr66-Gly67-, requires the native Protein fold for both formation and fluorescence emission. The structure of Thr65 GFP has been determined at 1.9 angstrom resolution. The Protein fold consists of an 11-stranded beta barrel with a coaxial helix, with the chromophore forming from the central helix. Directed mutagenesis of one residue adjacent to the chromophore, Thr203, to Tyr or His results in significantly red-shifted excitation and emission maxima.
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wavelength mutations and posttranslational autoxidation of Green Fluorescent Protein
Proceedings of the National Academy of Sciences of the United States of America, 1994Co-Authors: Roger Heim, Douglas Prasher, Roger Y TsienAbstract:Abstract The Green Fluorescent Protein (GFP) of the jellyfish Aequorea victoria is an unusual Protein with strong visible absorbance and fluorescence from a p-hydroxybenzylidene-imidazolidinone chromophore, which is generated by cyclization and oxidation of the Protein's own Ser-Tyr-Gly sequence at positions 65-67. Cloning of the cDNA and heterologous expression of Fluorescent Protein in a wide variety of organisms indicate that this unique posttranslational modification must be either spontaneous or dependent only on ubiquitous enzymes and reactants. We report that formation of the final fluorophore requires molecular oxygen and proceeds with a time constant (approximately 4 hr at 22 degrees C and atmospheric pO2) independent of dilution, implying that the oxidation does not require enzymes or cofactors. GFP was mutagenized and screened for variants with altered spectra. The most striking mutant fluoresced blue and contained histidine in place of Tyr-66. The availability of two visibly distinct colors should significantly extend the usefulness of GFP in molecular and cell biology by enabling in vivo visualization of differential gene expression and Protein localization and measurement of Protein association by fluorescence resonance energy transfer.
