Mutants of monomeric red fluorescent protein mRFP1 at residue 66: Structure modeling by molecular dynamics and search for correlations with spectral properties

Abstract

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...

Laser Fluorescence Spectroscopy: Application in Determining the Individual Photophysical Parameters of Proteins

“…It was predicted that the volume of the amino acid residue at position 66 can correlate with the optical characteristics of the mutants. The experimental results presented in this Section are consistent with the results of simulations performed by (Khrameeva et al, 2008). For the mutants with non-polar groups at residue 66 (alanine, leucine and phenylalanine) there is no correlation effect.…”

“…The deviations from chromophore coplanarity are responsible for the changes in the optical characteristics for mCherry and mStrawberry (Shu et al, 2006). The interrelation between the optical properties of the monomeric red FPs and the geometry of their chromophores was also confirmed by the molecular dynamics simulations conducted for mRFP1 mutants with single polar amino acid substitutions at position 66 (Khrameeva et al, 2008). The simulations have shown that the substitutions have an influence on the torsion angles in the phenolic and imidazolidine rings of the chromophore as well as on the torsion angles in the regions of connection between these rings and chromophore attachment to -barrel.…”

“…The seven mutants with substitution of the glutamine 66 for the serine (protein mRFP1/Q66S 2 ), cysteine (mRFP1/Q66C), asparagine (mRFP1/Q66N), histidine (mRFP1/Q66H), alanine (mRFP1/Q66A), leucine (mRFP1/Q66L) and phenylalanine (mRFP1/Q66F) have been created. The method for fabrication and purification of proteins is described in (Vrzheshch et al, 2008). All the experiments were performed in a 0.06 M phosphate buffer at a temperature of 25±1 °C.…”

“…7 (c, d)). These radicals, in turn, belong to the protein shell (the -barrel) and are rather rigidly bonded to it (Khrameeva et al, 2008). A change in the geometry of the side radical at position 66 will in this case cause a change in the geometry of the chromophore imidazolidine ring, because the interaction of the chromophore with the glutamine-42 and glutamine-213 through the hydrogen bond network can be distorted and a new bond with glutamate-215 can be formed.…”

Module based antibody engineering: A novel synthetic REDantibody

“…The excitation and emission wavelengths of REDantibody were determined to be 584nm and 607nm respectively (as shown in Figure 5A), are identical to that of mRFP (Campbell et al, 2002; Khrameeva et al, 2008), confirming that the fusing immunoglobulin domains to both ends of a FP did not compromise the fluorescent properties. Moreover, it may be possible to detect as little as 9.5 pmoles of REDantibody using a standard fluorometer as shown in figure 5B.…”