Reducing the Environmental Sensitivity of Yellow Fluorescent Protein

By O. Griesbeck, G. Baird, Robert E. Campbell, D. Zacharias, Roger Tsien. Journal of Biological Chemistry, 2001

1,050 citations recorded by the source corpus.

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 °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 °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 [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.

Subfields: Biophysics, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/1d2c0d80149085a1c00d6d85637919d9fcd3407d

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Citations in the corpus

A Novel Role for the TIR Domain in Association with Pathogen-Derived Elicitors (2007, methodology): “For this, we used improved EYFP and ECFP variants, Citrine and Cerulean, respectively, which produce greater fluorescence signal and hence allow easier detection of our fusion proteins [35,36].”
Fluorescent proteins and their applications in imaging living cells and tissues. (2010, background): “The universality and relative simplicity of such a design have led to the development of numerous FRETbased sensors of this type, including GES for monitoring changes in the concentration of Ca(2) (130, 153, 262, 263, 291, 292, 295, 304, 307, 327, 434), cyclic nucleotides (174, 317, 318, 375, 445, 5”
A guide to choosing fluorescent proteins (2005, background): “Early YFP variants were relatively chloride sensitive, a problem that has been solved in the Citrine and Venus (and likely YPet) variant”
Measuring single-cell gene expression dynamics in bacteria using fluorescence time-lapse microscopy (2011, background): “Motorized xy stage , such as an ASI MS2000 (13).”
Optimized cassettes for fluorescent protein tagging in Saccharomyces cerevisiae (2004, background): “Citrine is an improved version of YFP that has increased folding efficiency at high temperature, increased resistance to pH and Cl−, and increased photostability (Griesbeck et al., 2001; Miyawaki et al., 1999).”
Development of an optimized backbone of FRET biosensors for kinases and GTPases (2011, neutral): “…or donor were Venus (Nagai et al., 2002), cpVenus (cp50Venus, cp157Venus, cp173Venus, cp195Venus, cp229Venus; Nagai et al., 2004), mCitrine (Griesbeck et al., 2001), YPet, CyPet (Nguyen and Daugherty, 2005), mTFP1 (Ai et al., 2006), mTurquoise, mTurquoise-GL (Goedhart et al., 2010), ECFP,…”
Genetically encoded calcium indicators for multi-color neural activity imaging and combination with optogenetics (2013, methodology): “…CFP, and YFP have been published, often by mutating positions near the chromophore to improve folding, maturation, brightness, and photostability (Griesbeck et al., 2001; Nagai et al., 2002; Rizzo et al., 2004; Nguyen and Daugherty, 2005; Ai et al., 2006, 2007; Kremers et al., 2006, 2007; Mena…”
Acyl-Lipid Metabolism (2013, background): “New variants of YFP, Venus, and Citrine have improved pH and chloride sensitivity (Griesbeck et al., 2001; Nagai et al., 2002).”
PTRF-Cavin, a conserved cytoplasmic protein required for caveola formation and function. (2008, neutral): “( Griesbeck et al., 2001 ) and A206K (Zacharias et al., 2002).”