Visible fluorescent proteins properties

The most prominent feature of the chemistry of flavin is its redox properties. These properties make flavin especially suitable for its broad involvement in biological reactions. In the following the pH-dependent species formed in one- and two-electron reductions will be dealt with first, including their visible absorption and fluorescence properties. These physical properties form the basis of many kinetical and analytical studies. In Scheme 3 the structures refer to the free and protein-bound prosthetic groups (cf. Scheme 1). To study the physical properties of the flavocoenzymes often N(3)-alkylated lumiflavin (R = CH3) is used which is better soluble in a variety of solvents. Other physical and chemical properties of these species will be discussed subsequently. 

Direct labeling of a biomolecule involves the introduction of a covalently linked fluorophore in the nucleic acid sequence or in the amino acid sequence of a protein or antibody. Fluorescein, rhodamine derivatives, the Alexa, and BODIPY dyes (Molecular Probes [92]) as well as the cyanine dyes (Amersham Biosciences [134]) are widely used labels. These probe families show different absorption and emission wavelengths and span the whole visible spectrum (e.g., Alexa Fluor dyes show UV excitation at 350 nm to far red excitation at 633 nm). Furthermore, for differential expression analysis, probe families with similar chemical structures but different spectroscopic properties are desirable, for example the cyanine dyes Cy3 and Cy5 (excitation at 548 and 646 nm, respectively). The design of fluorescent labels is still an active area of research, and various new dyes have been reported that differ in terms of decay times, wavelength, conjugatibility, and quantum yields before and after conjugation [135]. New ruthenium markers have been reported as well [136]. 

As described above, the intrinsic fluorophores that nature provides in proteins generally have rather low absorption coefficients and quantum yields. For many applications, brighter fluorescence probes are needed, and emission in the visible region is desirable. One way to meet these needs is by labeling with a fluorescent dye. A wide range of fluorescence probes is now available for this purpose (see Fig. lb). A summary of the spectroscopic and photophysical properties of many fluorescence probes is available in References 5 and 6. 

Modem methods for study of metal-activated enzymes include NMR and ESR spectroscopy, water relaxation rates by pulsed NMR (PRR), atomic absorption, Mbssbauer, X-ray and neutron diffraction, high-resolution electron microscopy, UV/visible/IR spectroscopy, laser lanthanide pertubation methods, fluorescence, and equilibrium and kinetic binding techniques. Studies with Mg(II)-activated enzymes have been hampered by the lack of paramagnetic or optical properties that can be used to probe its environment, and the relative lack of sensitivity of other available methods initial velocity kinetics, changes in ORD/CD, fluorescence, or UV properties of the protein, atomic absorption assays for equilibrium binding, or competition with bound Mn(II) °. Recent developments in Mg and 0-NMR methodology have shown some promise to provide new insights . ... 

We have observed three types of effects on the structures of enzymes as the temperature is lowered in cryosolvents (1) no apparent change in the protein conformation, with the possible exception of decreased mobility of the surface side chains (2) conformational transitions, usually marked by little effect on the catalytic properties and (3) increased association of subunits. In most cases no detectable effects of decreasing temperature on the enzyme s structure have been detected by such procedures as monitoring the intrinsic fluorescence (16), or intrinsic visible absorbance in the case of flavin enzymes (Fink and... 

In current work the approach based on the simultaneous use of nonlinear laser fluorimetiy, spectrophotometry and conventional fluorimetry methods has been applied for investigation of the photophysical properties of the protein molecules of different complexity. The full set of photophysical p>arameters of the fluorophores (tryptophan residues) of human and bovine serum albumins has been determined. The photophysical processes in the spectral forms of the red FP mRFPl under UV (266 nm) and visible (532 nm) irradiation are described quantitatively. The individual photophysical parameters of the new mutants of the rtiRFTT protein (a single substitution at the 66 amino acid position) were determined. It was shown that the individual extinction coefficient of the red chromopore of the proteins correlate positively with the volume of the substituted amino acid residue at position 66 (for polar substitution). A similar correlation has been described for the position of the maximum of the absorption, fluorescence excitation and emission sp>ectra the position of the maximum moves to the red with increasing the volume of the residue. In addition, the partial concentration of the fluorescent spectral form in the resultant solution of each FP variant has been determined. 

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