Blue fluorescent proteins emission spectra

Spectral properties. The purified photoprotein is practically colorless, although its absorption spectrum (Fig. 7.1.4) shows a very slight absorption in the region of 330-380nm in addition to the 280 nm protein peak. The solution of photoprotein is moderately blue fluorescent, with an emission maximum at 453-455 nm and an... 

Figure 3.29 shows file fluorescence emission spectra of lO pM Quin-2 at different concentrations of calcium (0 to 40 pM) in the absence (A) and in the presence (B) ol 15.3 pM human serum albumin. From the data of figure 3.29, the authors generate steady-state emisaon spectra of the protein (Fig. 3.30a), Quin-2 (Fig. 3.30b), file Quin-2-calcium complex (Fig. 3.30c) and the Quin-2 - protein complex (Fig. 3.30d). One can notice that Quin-2 fluorescence emission spectrum in presence ofhuman serum albumin is blue slufted compared to Quin-2 flee in solution. The Quin-2-calcium complex is however red-shifted.

Human immunoefficiency virus (HIV) is the agent responsible for the acquired immunodeficiency syndrom (AIDS). HIV-1 protease is among the targets identified for chemotherapy. It is a homodimeric aspartyl protease of 99 amino acid residues per monomer. The protein contains two Trp residues (W6 and W42) per monomer located in different environment. The fluorescence emission spectrum of the protein shows a peak at 341 nm 215 nm) indicating that the Trp residues are responsible for the protein emission. Also, tlie position of this peak is blue-shifted compared to the fluorescence maximum of Tryptophan in water and thus the average exposure of the Trp residues to the solvent is not total. Trp residues are partly buried within the hydrophobic core of the protein. 

The shift of the peak intensity wavelength of the fluorescence emission spectrum intensity in Figure 8.1 also contains significant information about the protein layer adsorbed on the NP. The blue shift of this feature is symptomatic of a shift of the dielectric properties of the medium, or more specifically the polarity of the local environment of the emitter species with the observed blue shift corresponding to a relatively nonpolar environment. Evidently, the local dielectric environment within the fully developed adsorbed protein layers is less polar than the corresponding emitter environment of the protein dispersed in solution. This is another easily understood trend from a qualitative standpoint. 

The use of 1-dimethylamino-5-naphthyl sulphonate ("dansyl") as a label was first introduced in 1952 for the determination of the rotational diffusion of protein molecules from the anisotropy of the dansyl fluorescence (1). Somewhat later, (2) it was found that the emission intensity of fluorophores of this type is strongly increased when they are adsorbed from water solution on proteins This increase in quantum yield of emission (accompanied by a blue-shift of the emission spectrum) could be correlated with a decreasing polarity of the medium (3). The effect can be explained as follows If the dipolar interaction of the excited chromophore with the solvent is smaller than this interaction in the ground state, the emitted quantum will be increased at the same time, the probability of nonradiative deactivation will be reduced, increasing the quantum yield of fluorescence (4). If the fluorescent label can be attached to a well-defined site on a protein, its emission characteristics will "report" about the polarity of this site (5) and the use of... 

Naphthalene-sulphonate derivatives such as 8-anilino-l -naphthalene sulphonate (ANS) have an amphipathic character and, therefore, bind to phospholipid membranes, to detergent mice I Is and to hydrophobic sites on proteins (Slavik, 1982). The binding Is accompanied by a fluorescence enhancement and by a blue-shift of the emission spectrum due to the effect of the local apolar environment on the fluorescence yield of the bound dye. Cantley and Hammes (1976) reported that the fluorescence of ANS was enhanced upon binding to chloroplast coupling factor 1 (CF]). Neumann et al. (1979) have reported recently that Touloldlnonaphthalenesulphate (TNS) acts as an "energy transfer Inhibitor" of photophosphorylatlonc These results Indicate a specific Interaction of naphthalene sulphonates with CF]. 

The absorption spectrum of the photoprotein showed a small peak (Xmax 423 nm, with a shoulder at about 450 nm) in addition to the protein peak at 280nm (Fig. 10.1.2). The peak at 423nm decreased slightly upon the FI202-triggered luminescence reaction. The photoprotein is fluorescent in greenish-blue (emission A.max 482 nm), which coincides exactly with the luminescence spectrum of the photoprotein... 

Luminescence measurements on proteins occupy a large part of the biochemical literature. In what surely was one of the earliest scientific reports of protein photoluminescence uncomplicated by concurrent insect or microorganism luminescence, Beccari (64), in 1746, detected a visible blue phosphorescence from chilled hands when they were brought into a dark room after exposure to sunlight. Stokes (10) remarked that the dark (ultraviolet) portion of the solar spectrum was most efficient in generating fluorescent emission and identified fluorescence from animal matter in 1852. In general, intrinsic protein fluorescence predominantly occurs between 300 nm and 400 nm and is very difficult to detect visually. The first... 

The fluorescence intensity resolved by wavelength constitutes the fluorescence spectrum. The wavelengths of fluorescence photons contain information about the environment of the fluorophore and the sample heterogeneity. Por example, as described above, buried Trp residues tend to have blue-shifted emission bands (Xmax < 330 mn), whereas Trp residues partially or fully exposed to water have red-shifted emission bands ( max > 340 mn). Therefore, protein conformational changes or unfolding may be accompanied by shifts in the native fluorescence spectra. Pluorescence spectra can be measured on a standard fluorometer, which is available from many manufacturers. 

The absorption band at 384 nm is composed of contributions of the radical species and the second chromophore, whereas the fluorescence spectra with excitation maxima at 398 nm and emission maxima at 470-480 nm are attributed to the pterin alone (146, 155). The 7,8-dihydropterin cofactor, Xmax = 360 nm when free in solution and 390 nm when protein bound, is labile at neutral pH, readily decomposing upon denaturation to form products without significant visible absorption maxima. The photoreduction described above also reduces the second cofactor but in an irreversible manner with complete loss of its fluorescence and visible absorption characteristics (157). Reduction of the blue semiquinone FAD cofactor to the fully reduced form has no effect on the absorption spectrum of the pterin, suggesting that the absorption spectrum of the second cofactor must be independent of the oxidation state of the flavin and that the two cofactors are electronically isolated from each other (157). However, reduction of the flavin radical results in an increase in the fluorescence of the second cofactor, possibly indicating that the flavin radical acts as a potent quencher of fluorescence of the 7,8-dihydropterin. 

The measurements of the fluorescence emission spectra of the proteins (data not showed) revealed that the fluorescence of both proteins is blue shifted relative to the tryptophan fluorescence (353 nm) in a buffer solution (Banishev et al., 2008a). This is due to a decrease in the tryptophan environment polarity in the proteins. The maximum of the HSA fluorescence (332 nm) is blue shifted in comparison with BSA (342 nm). Since the fluorescence spectrum of the tryptophan residues reflects the polarity of their nearest environment, and since the properties of the environments of Trp>-212 in BSA and Trp-214 in HSA are similar (Eftink et al., 1977), such a shift can be related to the fact that BSA contains tryptophan Trp>-134 located in the environment with a higher polarity (in comparison with Trp)-212). Thus, the total fluorescence spectrum of BSA is red shifted. This result will be necessary for choosing the registration wavelength in measuring the acceptor and donor fluorescence when the nonlinear and kinetic curves will be measured (Section 6.1). 

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