GFP

We first supposed that the field radiated into the piece by the transducer is known, thanks to the Champ-Sons model. Then, the main approximation used consists in making far field assumptions in the beam defect interaction area. In the case of a focused transducer we assume that the incident wavefronts on the defect are plane. This is equivalent to say that the defect is located in or near the transducer focal area and that a defect located outside this zone does not cause a significant echo. In the case of planar contact transducer, the incident wavefronts on the defect are assumed to be spherical The incident field on the defect is therefore approximated by the product of a spatial function gfp,0,z)describing the amplitude distribution in the beam and a time-delayed waveform < ) ft) representing the plane or spherical propagation in the beam. The incident field on the defect can therefore be approximated for ... 

Antocatalytic oxidation of GFP amino acids leads to the chromophore shown on the left. The green fluorescence requires fnrtlier interactions of the chromophore witli otlier parts of die protein. 

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

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.

Structure of GFP and its chromophore. To study the chro-mophore of GFP, a sample of GFP was denatured by heating it at 90°C. It was digested with papain, and then a peptide containing the fluorophore was isolated and purified from the digested mixture. The structural study of the peptide has indicated that the chromophore of GFP is an imidazolone derivative shown below (Shimomura, 1979). This chromophore structure was confirmed later by Cody etal. (1993) in a hexapeptide isolated from GFP. It is intriguing that the structure of the GFP chromophore is a part of the structure of coelenterazine. 

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. 

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).

Quantum yield of luciferin. Various values of quantum yield have been reported for coelenterazine in the luminescence reaction catalyzed by Renilla luciferase 0.055 (Matthews et al., 1977a), 0.07 (Hart, et al., 1979), and 0.10-0.11 (with a recombinant form Inouye and Shimomura, 1997). The quantum yield is significantly increased in the presence of Renilla green fluorescent protein (GFP) see below. 

The spectra of the luminescence of coelenterazine catalyzed by recombinant Renilla luciferase in the presence and absence of Renilla GFP are shown in Fig. 4.6.3 (Lorenz et al., 1991). Note that the luminescence intensity at the emission peak is increased more than... 

Fig. 4.6.3 Bioluminescence emission spectra measured with coelenterazine plus 1 i.M Renilla luciferase in the absence (a) and presence (b) of 1 jlM Renilla GFP. From Lorenz et al., 1991.

Table 4.7.1 A Comparison of Aequorea GFP and Renilla GFP (Ward, 1998, modified)...

Cutler, M. W., and Ward, W. W. (1997). Spectral analysis and proposed model for GFP dimerization. In Hasting, J.W., et al. (eds.), Bioluminescence and Chemiluminescence Molecular Reporting with Photons, pp. 403-406. Wiley, New York. 

Gorokhovatsky, A. Y., et al. (2004). Fusion of Aequorea victoria GFP and aequorin provides their Ca2+-induced interaction that results in red shift of GFP absorption and efficient bioluminescence energy transfer. Biochem. Biophys. Res. Commun. 320 703-711. 

Kojima, S., et al. (1997). Mechanism of the redox reaction of the Aequorea green fluorescent protein (GFP). Tetrahedron Lett. 38 2875-2878. 

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... 

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