Cyan fluorescent proteins

Villoing A, Ridhoir M, Cinquin B, Erard M, Alvarez L, Vallverdu G, Pemot P, Grailhe R, Merola F, Pasquier H (2008) Complex fluorescence of the cyan fluorescent protein comparisons with the FU48D variant and consequences for quantitative cell imaging. Biochemistry 47 12483-12492... 

Chudakov DM, Verkhusha W, Staroverov DB, Souslova EA, Lukyanov S, Lukyanov KA (2004) Photoswitchable cyan fluorescent protein for protein tracking. Nat Biotechnol... 

Henderson JN, Remington SJ (2005) Crystal structures and mutational analysis of amFP486, a cyan fluorescent protein from Anemonia majano. Proc Natl Acad Sci USA 102 12712-12717... 

Ai HW, Henderson JN, Remington SJ, Campbell RE (2006) Directed evolution of a monomeric, bright and photostable version of Clavularia cyan fluorescent protein structural characterization and applications in fluorescence imaging. Biochem J 400 531-540... 

Malo GD, Wang M, Wu D, Stelling AL, Tonge PJ, Wachter RM (2008) Crystal structure and Raman studies of dsFP483, a cyan fluorescent protein from Discosoma striata. J Mol Biol 378 871-886... 

Ai HW, Olenych SG, Wong P, Davidson MW, Campbell RE (2008) Hue-shifted monomeric variants of Clavularia cyan fluorescent protein identification of the molecular determinants of color and applications in fluorescence imaging. BMC Biol 6 13. doi 10.1186/1741-2007-1186-1113... 

Rizzo MA, Springer GH, Granada B, Piston DW (2004) An improved cyan fluorescent protein variant useful for FRET. Nat Biotechnol 22 445 -49... 

Goedhart J, van Weeren L, Hink MA, Vischer NO, Jalink K, Gadella TWJ (2010) Bright cyan fluorescent protein variants identified by fluorescence lifetime screening. Nat Meth 7 137-139... 

The analysis of the histograms of photon arrival times is equivalent in both cases and relies on fitting appropriate model functions to the measured decay. The selection of the fitting model depends on the investigated system and on practical considerations such as noise. For instance, when a cyan fluorescent protein (CFP) is used, a multi-exponential decay is expected furthermore, when CFP is used in FRET experiments more components should be considered for molecules exhibiting FRET. Several thousands of photons per pixel would be required to separate just two unknown fluorescent... 

Cyan fluorescent proteins (CFPs) have blue-shifted excitation and emission spectra, because of the mutation Tyr66Trp inside the chromophore (Fig. 5.3C) [34], CFP fluorescence (Ex 435 nm/Em 474 nm) is less blue-shifted than for EBFP and CFP excitation is intermediate to the excitation of the neutral and anionic chromo-phores of avGFP [4], CFPs are widely used for dual-color imaging and FRET applications together with yellow fluorescent proteins (YFP, Section 3.6). 

Similarly to dyes, some fluorescent proteins can be incorporated into polymeric beads to be used as an alternative for ion sensing. For example, a reporter protein (composed of a phosphate-binding protein, a FRET donor (cyan fluorescent protein) and a FRET acceptor (yellow fluorescent protein)) was incorporated into polyacrylamide nanobeads by Sun et al. [46]. FRET was inhibited upon binding of phosphate. Kopelman and co-workers [47] used a similar approach to design a nanosensor for copper ions. They have found that fluorescence of red fluorescent protein DsRed (commonly used as a label) is reversibly quenched by Cu2+ and Cu+. Both DsRed and Alexa Fluor 488 (used as a reference) were entrapped into polyacrylamide nanobeads. Typically, up to 2 ppb of copper ions can be reliably measured. It should be mentioned, that in contrast to much more robust dyes, mild conditions upon polymerization and purification are very important for immobilization of the biomolecule to avoid degradation. 

A UV laser is needed for exciting the blue-fluorescing agents, 4, 6-diamidino-2-phenyhndole (DAPI) and Hoechst 33342, which are DNA-intercalating stains, and for indo-1, a fluorescent calcium chelator dye. Violet diode lasers that are offered in some newer instruments accommodate fluorochromes such as Cascade Blue, Pacific Blue, and cyan fluorescent protein, and are also capable of exciting DAPI (Shapiro and Perlmutter 2001 Telford et al., 2003). 

FRET requires the presence of two fluorophores, one with a shorter emission wavelength (donor) and another with a longer emission wavelength (acceptor). The fluorophores must be chosen such that there is sufficient overlap of the donor emission spectrum and the acceptor excitation spectrum. When FRET occurs, which requires the proximity of the two fluorophores, excitation of the donor results in transfer of energy to the acceptor and, hence, emission at the wavelength characteristic for the acceptor. FRET can be seen with various kinds of fluorophores, but most recendy it has been used in particular with variants of GFPs because this permits FRET in intact cells. The most frequently used pairs of GFPs are the cyan fluorescent protein (CFP) and the yellow fluorescent protein (YFP) variants. The donor CFP is excited at its maximum... 

DOTMA E. coli E EBV ECFP ECV EGFP ELISA EYFP FACS FdG FH2 FH4 FK506 FLP propane-aminium-trifluoracetate 7V-[2,3-(dioleyloxy) propyl]-/V,/V,/V-trimethyl ammonium chloride Escherichia coli erythromycine operon/repressor Epstein-Barr virus enhanced cyan fluorescence protein extracellular viral particles enhanced green fluorescence protein enzyme-linked immunosorbent assay enhanced yellow fluorescence protein fluorescence-activated cell sorter fluorescein di- 3-D-galactopyranoside dihydrofolate tetrahydrofolate human immunophilins native recombinase isolated from the 2pm plasmid from Saccharomyces cerevisiae... 

Table 4-1. CPU time for the perturbation selection. Cyan Fluorescent Protein, C H l C (Crsymmetry), with DZP level basis sets. The Is core and corresponding virtual orbitals were frozen. Total number of active space is 290 (51 occ. 239 unocc.)...

Volkmer A, Subramaniam V, Birch DJS, Jovin TM. One- and two-photon excited fluorescence lifetimes and anisotropy decays of green fluorescent proteins. Biophys. J. 2000 78 1589-1598. Subramaniam V, Hanley QS, Clayton AHA, Jovin TM. Photophysics of green and red fluorescent proteins implications for quantitative microscopy. Methods Enzymol. 2003 360 178-201. Rizzo MA, Springer GH, Granada B, Fdston DW. An improved cyan fluorescent protein variant useful for FRET. Nature Biotechnol. 2004 22 445-449. 

Further studies using green fluorescent protein or cyan fluorescent protein tagged receptors showed that a1B/a1D-AR heterodimerization appeared to completely control the surface expression and functional coupling of a1D-ARs on the plasma membrane (76). Coexpression of a1B, but not a1A-, ARs resulted in almost exclusively surface localization of the normally intracellular a1D-ARs, consistent with the specificity observed in previous coimmunoprecipitation studies. Further studies showed that the hydrophobic core of the a1B-AR is the major structural determinant of this interaction, and that G protein coupling was not required (76). These studies suggest that subtype-specific heterodimerization of ARs may control surface expression, and that these observations maybe relevant to many other class I G protein-coupled receptors, for which the functional consequences of this phenomenon are still poorly understood. 

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