title: Reducing the environmental sensitivity of yellow fluorescent protein. Mechanism and applications.
abstract: 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.
authors.affiliation: Howard Hughes Medical Institute, University of California, San Diego, La Jolla, California 92093-0647, USA.
authors.forename: O
authors.initials: O
authors.lastname: Griesbeck
authors.forename: G S
authors.initials: GS
authors.lastname: Baird
authors.forename: R E
authors.initials: RE
authors.lastname: Campbell
authors.forename: D A
authors.initials: DA
authors.lastname: Zacharias
authors.forename: R Y
authors.initials: RY
authors.lastname: Tsien
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism* / Q000528:radiation effects; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D002712:Chlorides / Q000494:pharmacology; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics; D005456:Fluorescent Dyes; D006056:Golgi Apparatus / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008164:Luminescent Proteins / Q000737:chemistry* / Q000378:metabolism* / Q000528:radiation effects; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010782:Photolysis; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection
chemical_list: D001426:Bacterial Proteins; D002147:Calmodulin; D002712:Chlorides; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D011994:Recombinant Proteins; C434479:citrine protein, bacteria; C054562:yellow fluorescent protein, Bacteria; D002118:Calcium