Förster resonance energy transfer (FRET) is a powerful tool to investigate biochemical and biophysical processes in
vitro and in vivo. We present the computational study of a novel FRET system, namely, a fuse protein that is composed of two far-red fluorescent proteins FusionRed and eqFP670 joined with a linker (FusionRed-Linker1-eqFP670). The latter contains a tetrapeptide DEVD motif which can be specifically cleaved by caspase-3. FusionRed acts as an energy
donor and eqFP670 as an energy acceptor in this pair. We carried out the comprehensive study of the factors that
influence FRET efficiency, including, in particular, distances between donor and acceptor, as well as relative orientations of transition dipole moments from donor to acceptor and the structure of linker between donor and acceptor.
We started from the primary structure of the proteins and also used available data on the similar β-barrels to construct the full-atom 3D structure of FusionRed-Linker1-eqFP670 sensor. We used a protein-protein docking procedure to construct the tetramer structure of the fuse protein. We compared this sensor with another FLIM/FRET-based sensor on caspase-3, TagRFP-23-KFP and explained the differences in their properties.
KEYWORDS: Chromophores, Proteins, Systems modeling, Fluorescent proteins, Absorption, Green fluorescent protein, Biomedical optics, Crystals, Molecules, Chemical species
Modern computational approaches based on quantum mechanical methods to characterize structures and optical spectra
of biological chromophores in proteins are intensively used to gain knowledge of events occurring upon of their photoexcitation.
Primary attention is paid to the species from the family of the green fluorescent protein applied as
biomarkers in living cells. We apply quantum chemical approaches for accurate calculations of the structures of the
chromophore binding pockets and to estimate spectral bands corresponding to the S0-S1 optical transitions of the intriguing kindling protein asFP595. Its precursor, the chromoprotein asCP from the sea anemony Anemonia sulcata is
characterized by distinctive spectral properties: at low light intensities the wild-type protein is weakly fluorescent with
the very low quantum yield, however, high intensity irradiation with green light leads to a drastic increase of quantum
yield. This phenomenon is now termed "kindling". In simulations, the model system is designed as a molecular cluster
constructed on the basis of available crystal structures of the related protein. The equilibrium geometry of the cluster is
optimized using density functional theory approximations. The vertical excitation energies corresponding to the S0-S1 transitions are computed by using the semiempirical ZINDO technique. A special attention is paid to evaluate effects of
point mutations in the vicinity of the chromophore group. Theoretical data provide important information on the
chromophore properties aiming to interpret the results of experimental studies and applications of this fluorescent protein.
Modern molecular modeling tools are intensively used to gain knowledge of events occurring upon photoexcitation of
organic chromophores in the gas-phase, in solution and in protein matrices. We applied quantum mechanical approach
to estimate equilibrium geometry configurations as well as positions and intensities of spectral bands for a number of
red fluorescent proteins, including the DsRed from Discosoma coral, and its mutants of the so-called mFruits series. As
demonstrated in our previous simulations for GFP and blue fluorescent proteins, this strategy was proven to be
productive for modeling. The model system is designed as a molecular cluster constructed on the basis of available
crystal structures of the related protein. The equilibrium geometry of the cluster is optimized using density functional
theory approximations. The vertical excitation energies corresponding to the S0-S1 transitions are computed by using the
semiempirical ZINDO technique. Mechanisms of photoexcitation, identification of the functional states of the
chromophores, elucidation the role of point mutations in the photoreceptor proteins are considered on the basis of
simulations.
KEYWORDS: Chromophores, Green fluorescent protein, Fluorescent proteins, Proteins, Systems modeling, Chemical analysis, Chemical species, Computational modeling, Biomedical optics, Chemistry
Modern computational approaches based on quantum mechanical methods to characterize structures and optical spectra
of biological chromophores in the gas phase, in solutions and proteins are discussed. Primary attention is paid to the
chromophores from the family of the green fluorescent protein (GFP) widely used as a biomarker in living cells.
Beyond GFP, photophysical properties of the monomeric teal fluorescent protein (mTFPI) and the kindling fluorescent
protein asFP595 are simulated. We apply modern quantum chemical approaches for high level calculations of the
structures of the chromophore binding pockets and to estimate spectral bands corresponding to the S0-S1 optical
transitions. A special attention is paid to evaluate effects of point mutations in the vicinity of the chromophore group.
Theoretical data provide important information on the chromophore properties aiming to interpret the results of
experimental studies of fluorescent proteins.
We report the results of quantum mechanical - molecular mechanical (QM/MM) simulations aiming to elucidate the mechanism of kindling of the initially non-fluorescent protein asFP595, which is a mutated variant of the chromoprotein asCP from the sea anemone Anemonia sulcata. asFP595 becomes brightly fluorescent (kindles) with emission at 595 nm in response to intense light irradiation at 568 nm. In simulations, we use the flexible effective fragment QM/MM method with the complete active space self-consistent field (CASSCF) wavefunctions in the quantum part and the AMBER force field parameters in the molecular mechanical part. We analyze the computed scans over potential energy surfaces of the ground and excited electronic states and consider details of the working hypothesis that the trans-cis isomerization of the chromophore group inside the protein is responsible for kindling.
Computational approaches to describe optical spectra of biological chromophores in proteins, in solutions and in the gas phase are discussed. Recently, accurate measurements of spectral properties for the series of chromophores in different media allowed the authors to estimate the positions of the bands with a high accuracy and to challenge theoreticians by stating that the measured S0-S1 transition wavelengths may be used as new benchmark values for the theory. The novel computational approaches based on the multiconfigurational quasidegenerate perturbation theory present the practical means how to adapt the high level methodology for calculations of accurate excitation energies in large biological chromophores. The theory is illustrated for a series of model compounds for which experimental data are available: the retinal molecule in the protonated Shiff-base form, the chromophores from the Green Fluorescent Protein family including the kindling protein asFP595, and the chromophore from the BLUF domain containing photoreceptor proteins.
We address the problem of quantitative evaluation of the absorption S0-S1 peaks &lgr; max of biological chromophores in
vacuo by using the state-of-art computational methods of quantum theory. In particularly, we rely on the second order
multiconfigurational quasidegenerate perturbation theory (MCQDPT2) following the complete active space selfconsistent
field (CASSCF) calculations. The use of augmented effective Hamiltonian operators in the MCQDPT2
framework allows us to correct deficiencies of the standard multistate approaches and to obtain stable saturated
solutions for the target low-lying excited states. A high accuracy of the proposed methodology is illustrated for several
photoactive protein chromophores in the gas phase including all-trans retinal, green fluorescent protein type
chromophores and photoactive yellow protein chromophores. It is shown that our approach provides correct ordering of
states and predicts maxima of absorption bands for the S0-S1 transitions within only a few nanometers from
experimental data.
KEYWORDS: Chromophores, Proteins, Chemical species, Crystals, Systems modeling, Chemistry, Fluorescent proteins, Quantum physics, Quantum computing, Green fluorescent protein
We present the results of modeling properties of the chromophore, 2-acetyl-4-(p-hydroxybenzylidene)-1-methyl-5-
imidazolone (AHBMI), from the newly discovered fluorescent protein asFP595 inside the protein environment by using
the combined quantum mechanical - molecular mechanical (QM/MM) method. In this approach, the chromophore unit
and the side chains of the nearest amino acid residues are assigned to the quantum subsystem. The starting coordinates
of heavy atoms were taken from the relevant crystal structures of the protein. Hydrogen atoms were added manually,
and the structure of the model protein system was optimized by using QM/MM energy minimization for the trans-form
of the chromophore. The Hartree-Fock/6-31G quantum chemical approximation and the AMBER force field parameters
were employed in geometry optimization. The points on potential energy surfaces of the ground and first and second
excited electronic states were computed with the complete active space self-consistent field approximation in the
quantum subsystem under different choices of the QM/MM partitioning. Possible pathways for the trans-cis photo
isomerization presumably responsible for the kindling properties of asFP595 as well as other mechanisms of photo
excitation are discussed.
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