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

Yellow mutants of the green fluorescent protein (YFP) are crucial constitue nts of genetically encoded indicators of signal transduction and fusions to monitor protein-protein interactions. However, previous YFPs show excessiv e pH sensitivity, chloride interference, poor photostability, or poor expre ssion 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 photost ability 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 crys tal structure of Citrine, showing that the methionine side chain fills what was once a large halide-binding cavity adjacent to the chromophore. Insert ion of calmodulin within Citrine or fusion of cyan fluorescent protein, cal modulin, a calmodulin-binding peptide and Citrine has generated improved ca lcium indicators. These chimeras can be targeted to multiple cellular locat ions 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 s ensitivity is desired and is particularly advantageous within genetically e ncoded fluorescent indicators of physiological signals.