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      A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum

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          Abstract

          Despite the existence of fluorescent proteins spanning the entire visual spectrum, the bulk of modern imaging experiments continue to rely on variants of the green fluorescent protein derived from Aequorea victoria. Meanwhile, a great deal of recent effort has been devoted to engineering and improving red fluorescent proteins, and relatively little attention has been given to green and yellow variants. Here we report a novel monomeric yellow-green fluorescent protein, mNeonGreen, which is derived from a tetrameric fluorescent protein from the cephalochordate Branchiostoma lanceolatum. This fluorescent protein is the brightest monomeric green or yellow fluorescent protein yet described, performs exceptionally well as a fusion tag for traditional imaging as well as stochastic single-molecule superresolution imaging, and is an excellent FRET acceptor for the newest generation of cyan fluorescent proteins.

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          Most cited references31

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          The green fluorescent protein.

          R Tsien (1998)
          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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            I-TASSER: a unified platform for automated protein structure and function prediction.

            The iterative threading assembly refinement (I-TASSER) server is an integrated platform for automated protein structure and function prediction based on the sequence-to-structure-to-function paradigm. Starting from an amino acid sequence, I-TASSER first generates three-dimensional (3D) atomic models from multiple threading alignments and iterative structural assembly simulations. The function of the protein is then inferred by structurally matching the 3D models with other known proteins. The output from a typical server run contains full-length secondary and tertiary structure predictions, and functional annotations on ligand-binding sites, Enzyme Commission numbers and Gene Ontology terms. An estimate of accuracy of the predictions is provided based on the confidence score of the modeling. This protocol provides new insights and guidelines for designing of online server systems for the state-of-the-art protein structure and function predictions. The server is available at http://zhanglab.ccmb.med.umich.edu/I-TASSER.
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              Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein.

              Fluorescent proteins are genetically encoded, easily imaged reporters crucial in biology and biotechnology. When a protein is tagged by fusion to a fluorescent protein, interactions between fluorescent proteins can undesirably disturb targeting or function. Unfortunately, all wild-type yellow-to-red fluorescent proteins reported so far are obligately tetrameric and often toxic or disruptive. The first true monomer was mRFP1, derived from the Discosoma sp. fluorescent protein "DsRed" by directed evolution first to increase the speed of maturation, then to break each subunit interface while restoring fluorescence, which cumulatively required 33 substitutions. Although mRFP1 has already proven widely useful, several properties could bear improvement and more colors would be welcome. We report the next generation of monomers. The latest red version matures more completely, is more tolerant of N-terminal fusions and is over tenfold more photostable than mRFP1. Three monomers with distinguishable hues from yellow-orange to red-orange have higher quantum efficiencies.
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                Author and article information

                Journal
                101215604
                32338
                Nat Methods
                Nat. Methods
                Nature methods
                1548-7091
                1548-7105
                17 October 2013
                24 March 2013
                May 2013
                01 November 2013
                : 10
                : 5
                : 10.1038/nmeth.2413
                Affiliations
                [1 ]Department of Photobiology and Bioimaging, The Scintillon Institute, 6404 Nancy Ridge Dr., San Diego, CA 92121
                [2 ]Allele Biotechnology and Pharmaceuticals, Inc., 6404 Nancy Ridge Dr., San Diego, CA 92121
                [3 ]Author to whom correspondence should be addressed
                [4 ]National High Magnetic Field Laboratory, 1800 E. Paul Dirac Dr., The Florida State University, Tallahassee, Florida 32310
                [5 ]Department of Biological Science, 1800 E. Paul Dirac Dr., The Florida State University, Tallahassee, Florida 32310
                [6 ]Department of Cellular & Integrative Physiology, Indiana University School of Medicine, 635 Barnhill Drive, MS 333, Indianapolis, IN 46202
                [7 ]Department of Microbiology, Tumor and Cell Biology (MTC), Karolinska Institutet, Box 280, SE-171 77 Stockholm, Sweden
                Article
                NIHMS516709
                10.1038/nmeth.2413
                3811051
                23524392
                07efe672-b5c8-4e57-97f1-0bb7988d08d0

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                History
                Funding
                Funded by: National Institute of Diabetes and Digestive and Kidney Diseases : NIDDK
                Award ID: R01 DK043701 || DK
                Categories
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                Life sciences
                Life sciences

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