The red fluorescent protein, DsRed, recently cloned from coral Discosoma sp. has one of the longest fluorescence waves and one of the most complex absorbance spectra among the family of fluorescent proteins. In this work we found that with time DsRed fluorescence decreases under mildly acidic conditions (pH 4.0^4.8) in a pH-dependent manner, and this fluorescence inactivation could be partially recovered by subsequent re-alkalization. The DsRed absorbance and circular dichroism spectra under these conditions revealed that the fluorescence changes were caused by denaturation followed by partial renaturation of the protein. Further, analytical ultracentrifugation determined that native DsRed formed a tight tetramer under various native conditions. Quantitative analysis of the data showed that several distinct states of protein exist during the fluorescence inactivation and recovery, and the inactivation of fluorescence can be caused by protonation of a single ionogenic group in each monomer of DsRed tetramer. ß
The red fluorescent protein DsRed recently cloned from Discosoma coral, with its significantly red-shifted excitation and emission maxima (558 and 583 nm, respectively), has attracted great interest because of its spectral complementation to other fluorescent proteins, including the green fluorescent protein and its enhanced mutant EGFP. We demonstrated that the much slower DsRed fluorescence development could be described by a three-step kinetic model, in contrast to the fast EGFP maturation, which was fitted by a one-step model. At pH below 5.0 DsRed fluorescence gradually decreased, and the rate and degree of this fluorescence inactivation depended on the pH value. The kinetics of fluorescence inactivation under acidic conditions was fitted by a two-exponential function where the initial inactivation rate was proportional to the fourth power of proton concentration. Subsequent DsRed alkalization resulted in partial fluorescence recovery, and the rate and degree of such recovery depended on the incubation time in the acid. Recovery kinetics had a lag-time and was fitted minimally by three exponential functions. The DsRed absorbance and circular dichroism spectra revealed that the fluorescence loss was accompanied by protein denaturation. We developed a kinetic mechanism for DsRed denaturation that includes consecutive conversion of the initial state of the protein, protonated by four hydrogen ions, to the denatured one through three intermediates. The first intermediate still emits fluorescence, and the last one is subjected to irreversible inactivation. Because of tight DsRed tetramerization we have suggested that obligatory protonation of each monomer results in the fluorescence inactivation of the whole tetramer.
A steady-state approximation of the generalized two-dimensional model of a bifunctional enzyme catalyzing independent proceeding of two one-pathway reactions is considered in a case of mutual influence of the active sites. Coexistence of fast and slow catalytic cycles in the reaction mechanism is analyzed. Conditions when the hierarchy of fast and slow catalytic cycles allows simplification of a two-dimensional model and its reduction to the one-dimensional cyclic schemes were determined. Kinetic equations describing these simplified schemes are presented.
Fluorescent proteins are widely used as markers for visualization of processes in intact biological systems. The family of fluorescent proteins includes proteins of various representatives of Coelenterata that are able to absorb light and emit in the visible spectral range. All flu orescent proteins have similar structure represented by 11 strand β barrel with α helix inside, which contains a chromophore in the middle. The chromophore is a het erogroup formed by an autocatalytic posttranslational reaction between three adjacent amino acid residues. The presence of the chromophore imparts color to the protein and induces its possible fluorescence [1].Fluorescence of proteins of this family is influenced by external conditions: pH [2,3], temperature [1], and ionic content of the medium [4,5], but structures of the chromophore and its nearest environment [1,6] are the key factors. The representatives of this family in matura tion rate, stability, spectral properties (absorption and flu orescence spectra, fluorescence quantum yield, photo bleaching, etc.) are described in [1,7]. Theoretical, experimental, and computational studies are carried out to investigate the mechanism of fluorescence of these proteins [8 11].Green fluorescent protein (GFP) was discovered in the early 1960s [12], but its active study began only after cloning of the GFP gene in 1992 [13] and demonstration of its heterologic expression in other organisms. In 1999, another family of colored proteins including the red pro tein DsRed was cloned from corals [14]. However, DsRed is a tetramer, and its use as a fluorescent marker is limit ed because of possible effects of its large molecular mass ISSN 0006 2979, Biochemistry (Moscow), 2008, Vol. 73, No. 10, pp. 1085 1095. © Pleiades Publishing, Ltd., 2008. Original Russian Text © E. E. Khrameeva, V. L. Drutsa, E. P. Vrzheshch, D. V. Dmitrienko, and P. V. Vrzheshch, 2008, published in Biokhimiya, 2008, Vol. 73, No. 10, pp. 1355 1367. Originally published in Biochemistry (Moscow) On Line Papers in Press, as Manuscript BM08 038, September 21, 2008 1085 Abbreviations: DsRed) red fluorescent protein drFP583 from coral Discosoma sp.; GFP) green fluorescent protein from Aequorea victoria; MD) molecular dynamics; mRFP1) monomeric red fluorescent protein, mutant of DsRed; Q66X) mutants of mRFP1 with replacement of residue 66 by residue X. * To whom correspondence should be addressed. Abstract-To study the interrelation between the spectral and structural properties of fluorescent proteins, structures of mutants of monomeric red fluorescent protein mRFP1 with all possible point mutations of Glu66 (except replacement by Pro) were simulated by molecular dynamics. A global search for correlations between geometrical structure parameters and some spectral characteristics (absorption maximum wavelength, integral extinction coefficient at the absorption maximum, excitation maximum wavelength, emission maximum wavelength, and quantum yield) was performed for the chromophore and its 6 A environment in mRFP1, Q66A, Q...
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