Yellow mutants of the green fluorescent protein (YFP) are crucial constituents of genetically encoded indicators of signal transduction and fusions to monitor proteinprotein interactions. However, previous YFPs show excessive pH sensitivity, chloride interference, poor photostability, or poor expression at 37°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,

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... wice the photostability of previous YFPs, and much better expression at 37°C and in organelles. The halide resistance is explained by a 2.2-Å 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. Yellow fluorescent proteins (YFPs) 1 were created (1) by mutating Thr 203 of the Aequorea victoria green fluorescent protein reticulum; PCR, polymerase chain reaction; MOPS, 4-morpholinepropanesulfonic acid; GT, galactosyltransferase.