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キーワード:
resonance energy-transfer
living cells
endoplasmic-reticulum
live
cells
in-vivo
ca2+
variants
ph
indicator
binding
Biochemistry & Molecular Biology
要旨:
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 degreesC. 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 degreesC and in organelles. The halide resistance is explained by a 2.2-Angstrom 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 [Ca2+] 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.
