Reducing the environmental sensitivity of yellow fluorescent protein. Mechanism and applications - PubMed (original) (raw)
. 2001 Aug 3;276(31):29188-94.
doi: 10.1074/jbc.M102815200. Epub 2001 May 31.
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- PMID: 11387331
- DOI: 10.1074/jbc.M102815200
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Reducing the environmental sensitivity of yellow fluorescent protein. Mechanism and applications
O Griesbeck et al. J Biol Chem. 2001.
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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.
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