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      Reducing the environmental sensitivity of yellow fluorescent protein. Mechanism and applications.

      The Journal of Biological Chemistry

      metabolism, Transfection, radiation effects, Binding Sites, Calcium, Calmodulin, chemistry, Chlorides, pharmacology, Cloning, Molecular, Crystallography, X-Ray, Escherichia coli, genetics, Fluorescent Dyes, Golgi Apparatus, HeLa Cells, Humans, Hydrogen-Ion Concentration, Luminescent Proteins, Models, Molecular, Mutagenesis, Site-Directed, Photolysis, Polymerase Chain Reaction, Protein Conformation, Recombinant Proteins, Amino Acid Substitution, Bacterial Proteins

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          Yellow mutants of the green fluorescent protein (YFP) are crucial constituents of genetically encoded indicators of signal transduction and fusions to monitor protein-protein interactions. However, previous YFPs show excessive pH sensitivity, chloride interference, poor photostability, or poor expression at 37 degrees C. Protein evolution in Escherichia coli has produced a new YFP named Citrine, in which the mutation Q69M confers a much lower pK(a) (5.7) than for previous YFPs, indifference to chloride, twice the photostability of previous YFPs, and much better expression at 37 degrees 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(2+)] 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.

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