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Aequorea GFP

Properties of GFP. Aequorea GFP is a relatively stable protein with a molecular weight of about 27,000 (Shimomura, 1979). The... [Pg.129]

Fig. 4.1.16 Luminescence spectrum of aequorin triggered by Ca2+ (solid line /.max 465 nm), and the fluorescence spectra of Aequorea GFP excitation (dashed line A.max 400 nm and 477 nm) and emission (dash-dot line 7max 509 nm). The dotted line is the fluorescence excitation spectrum of GFP in the light organs, showing that 480 nm excitation peak is almost missing — an evidence showing that GFP in light organs exists in an aggregated form having a very low E value at 480 nm. Fig. 4.1.16 Luminescence spectrum of aequorin triggered by Ca2+ (solid line /.max 465 nm), and the fluorescence spectra of Aequorea GFP excitation (dashed line A.max 400 nm and 477 nm) and emission (dash-dot line 7max 509 nm). The dotted line is the fluorescence excitation spectrum of GFP in the light organs, showing that 480 nm excitation peak is almost missing — an evidence showing that GFP in light organs exists in an aggregated form having a very low E value at 480 nm.
The gene of Aequorea GFP was cloned by Prasher et al. (1992), and expressed in E. coli and Caenorhabditis elegans by Chalfie et al. (1994) and in E. coli by Inouye and Tsuji (1994a). The X-ray structure of recombinant GFP was solved by Ormo et al. (1996) and Yang et al. (1996,1997). The protein is in the shape of a cylinder consisting of 11 strands of (3-sheets and an a-helix inside (which contains the chromophore), with short helical segments on the ends of the cylinder. Thus the chromophore is sealed and protected from the outside medium. [Pg.131]

Fig. 4.1.17 Graphic illustration of Forster-type resonance energy transfer from aequorin to Aequorea GFP. In the vessel at left, a solution contains the molecules of aequorin and GFP randomly distributed in a low ionic strength buffer. The vessel at right contains a solution identical with the left, except that it contains some particles of DEAE cellulose. In the solution at right, the molecules of aequorin and GFP are coadsorbed on the surface of DEAE particles. Upon an addition of Ca2+, the solution at left emits blue light from aequorin (Xmax 465 nm), and the solution at right emits green light from GFP (Xmax 509 nm). Fig. 4.1.17 Graphic illustration of Forster-type resonance energy transfer from aequorin to Aequorea GFP. In the vessel at left, a solution contains the molecules of aequorin and GFP randomly distributed in a low ionic strength buffer. The vessel at right contains a solution identical with the left, except that it contains some particles of DEAE cellulose. In the solution at right, the molecules of aequorin and GFP are coadsorbed on the surface of DEAE particles. Upon an addition of Ca2+, the solution at left emits blue light from aequorin (Xmax 465 nm), and the solution at right emits green light from GFP (Xmax 509 nm).
Table 4.7.1 A Comparison of Aequorea GFP and Renilla GFP (Ward, 1998, modified)... Table 4.7.1 A Comparison of Aequorea GFP and Renilla GFP (Ward, 1998, modified)...
Roth, A. F., and Ward, W. W. (1983). Conformational stability after protease treatment in Aequorea GFP. Photochem. Photobiol. 37S S71. [Pg.430]

Acantbephyra, 162, 336 Acantboscina, 336 Acholoe, 335 Achromobacter, 35, 36 Acorn worms (enteropneusts), 315 Acylhomoserine lactone, 43 Advice to students, 375 Aequorea, 159, 161, 162, 334, 375 Aequorea aequorea, 92-94, 346 collection, 93, 94 distribution, 92 squeezate, 94 synonyms, 92 Aequorea GFP, 150-154 chromophore, 153 cloning, 154 crystallization, 130 fluorescence quantum yield, 152 isolation, 129 molecular weight, 152 spectral properties, 130, 152 Aequorea victoria, 92 Aequorin, 92-129, 159, 160,172,173, 175, 346, 349, 350, 364, 375 assay, 98... [Pg.455]

Later on similar color shifts were found in the related coelenterates Obelia (a hydroid) and Renilla (a sea pansy) and it was suggested that the in-vivo excitation mechanism for coelenterate GFPs is based on radiationless energy transfer [3]. The first prove for this assumption was provided by Morise and coworkers in 1974 [4], They managed to purify and crystallize Aequorea GFP, and to demonstrate the efficient luminescence energy transfer between co-adsorbed Aequorin and GFP. In addition, the absorbance spectrum and fluorescence quantum yield was measured. [Pg.4]

Until 1999 Renilla GFP was the only biochemically well characterized fluorescent protein besides the Aequorea GFP. It proved to have a much higher extinction coefficient, resistance to pH-induced conformational changes and denaturation, and tendency to dimerize compared to Aequorea GFP [7]. However, chromophores from Aequorea and Renilla GFPs was shown to be chemically identical [7-9], indicating that the fluorescence properties of the proteins are depending on both, the chromophore and the corresponding environment provided by the amino-acid backbone. [Pg.5]

Until 1999 the Aequorea GFP remained the only cloned gene encoding a fluorescent protein. All the different GFP isoforms developed and used in molecular biology are derivatives of the GFP 10 cDNA cloned by Prasher and co-workers. Attempts to clone the gene for Renilla-GFP, which is supposed to be several times brighter than the Aequorea-GFP, were not successful. [Pg.6]

Up to now pointmutations substituting amino-acid residues of the chromophore leading to altered spectral properties have only be described for Aequorea GFP. However, mutation located outside the chromophore can also change the spectra of fluorescent protein, indicating that the chromophore environment provided by the B-can structure of the protein is of equal importance, Fig. (5). [Pg.15]

The proteins of this class and especially its first member, the Aequorea GFP are by for the best understood fluorescent proteins,... [Pg.17]

For the majority of GFPs knowledge about the mechanisms and processes responsible for the fluorescence of the proteins is lacking. However, in this respect the Aequorea GFP and its derivatives were investigated intensively and certainly can serve as model for the other proteins [48,49]. [Pg.19]

Ex excitation, Em emission, EC extinction coefficient, QY quantum yield, n.d. not determined Recombinant isoforms of Aequorea GFP are described in table 2. [Pg.20]

Mature Aequorea GFP as well as its recombinant derivatives are known to be highly soluble [51]. Formation of protein aggregates has only been seen for the wildtype Aequorea GFP [19]. In GFP... [Pg.29]

All blue fluorescent proteins known today are derivatives of Aequorea GFP. The first member of these class of proteins was obtained by intentional introduction of a single point mutation... [Pg.31]

As mentioned above BFPs are mutant isoforms of the Aequorea GFP carrying a histidine residue at position 66. In consequence, an imidazole is placed in the chromophore [52] causing a shift of the excitation and emission peaks towards shorter wavelengths. Further work led to the isolation of improved BFP isoforms with slightly higher molar extinction coefficient and quantum yield (increasing... [Pg.32]

Due to their extremely close relationship to GFP it is highly likely that these proteins share the weak dimerization tendency with its ancestor Aequorea GFP [19]. Although experimental evidence is lacking it has to be assumed that the introduction of mutations blocking the dimerization of GFP (Phe223Arg, Leu221Lys or Ala206Lys) will also be beneficial for GFP-derived BFPs. [Pg.34]

The first described member of the cyan fluorescent proteins (CFPs) resulted from a rationally designed chromophore mutation of Aequorea GFP. Heim and co-worker replaced Tyr66 with Trp and found the peak wavelength for excitation and emission of this GFP derivative (GFP-Y66W) to be shifted to 436 and 476 nm, respectively [52], Because of this blue-green/cyan light emission the protein was called cyan fluorescent protein or CFP. [Pg.35]

When it comes to protein aggregation the known GFP derived CFPs should behave just like Aequorea GFP with the mature proteins being highly soluble not giving rise to significant protein... [Pg.40]

Together with the protein classes BFP and CFP, also the first yellow fluorescent proteins (YFPs) were originally derived form Aequorea GFP by introduction of amino-acid exchanges at positions 65 (the first amino-acid of the chromophore) and 203 [51]. In fact, these mutants were rationally designed based on crystal structure of GFP in order to create a fluorescent protein sufficiently different in its fluorescence properties to allow for double-labeling experiments, FRET and other experimental applications. [Pg.41]

For YFPs derived from the Aequorea GFP (table 6) it has to be expected that they also do suffer from the thermosensitivity effect described for GFP. Indirect evidence is provided by the fact that the point-mutations described to improve the folding of GFP are also helpful for the GFP derived YFPs (see below). However, no direct evidence is present in published literature. [Pg.42]

For the GFP-derived YFPs it is, again, likely that they share the weak dimerization tendency of Aequorea GFP. As is true for the BFPs and CFP the introduction of mutations blocking the dimerization of GFP (F223R, L221K or A206K) should also be beneficial for YFPs. Nevertheless experimental data are lacking. Analysis of zoanYFP-II (see table 7) proved the tetramerization of this protein. [Pg.46]

Unlike Aequorea GFP and its derivatives the folding/maturation of DsRED does not suffer from thermosensitivity (Jach, unpublished results). However, while working with this protein it quickly turned out that the maturation-rate of this protein is extremely slow. At room-temperature the wildtype DsRED needs 24-36 hours to gain maximum fluorescence [28]. For HcRED maturation rates comparable to GFP have been published. For the other naturally occurring RFPs (see table 8) no such data are available yet. [Pg.51]

Wang Y, Wang G, O Kane DJ, Szalay AA. A study of protein protein interactions in living cells using luminescence resonance energy transfer (LRET) from Renilla luciferase to Aequorea GFP. Mol Gen Genet 2001 264 578-587. [Pg.110]


See other pages where Aequorea GFP is mentioned: [Pg.130]    [Pg.130]    [Pg.131]    [Pg.152]    [Pg.152]    [Pg.153]    [Pg.154]    [Pg.551]    [Pg.32]    [Pg.32]    [Pg.5]    [Pg.6]    [Pg.8]    [Pg.18]    [Pg.18]    [Pg.19]    [Pg.19]    [Pg.19]    [Pg.21]    [Pg.28]    [Pg.29]   
See also in sourсe #XX -- [ Pg.17 , Pg.18 , Pg.19 , Pg.20 , Pg.31 , Pg.32 ]




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