Fluorescent Protein asFP595 Research Articles

Modeling Structures and Spectra of Fluorescent Proteins in the Coordinate-Locking Cluster Approach: Application to the Photoswitchable Protein AsFP595

An interest in the fluorescent protein asFP595 is due to unexplained puzzles in its photophysical behavior. We report the results of calculations of structures, absorption, and emission bands in asFP595 by considering model molecular clusters in the coordinate-locking scheme. Both trans and cis conformations of the anionic chromophore are considered. Equilibrium geometry coordinates on the ground potential energy surface were optimized in the density functional theory approaches by considering both large- and reduced-size clusters. The cluster size was reduced to locate positions of the minimum energy points on the excited-state potential surface by using the configuration interaction singles approach. Vertical excitation energies and oscillator strengths were computed by using the ZINDO method. We show that consideration of large clusters mimicking the protein-containing pocket is an essential issue to calculate positions of absorption and emission bands with the accuracy compatible to experiments.

Modeling Photoabsorption of the asFP595 Chromophore

The fluorescent protein asFP595 is a promising photoswitchable biomarker for studying processes in living cells. We present the results of a high level theoretical study of photoabsorption properties of the model asFP595 chromophore molecule in biologically relevant protonation states: anionic, zwitterionic, and neutral. Ground state equilibrium geometry parameters are optimized in the PBE0/(aug)-cc-pVDZ density functional theory approximation. An augmented version of multiconfigurational quasidegenerate perturbation theory (aug-MCQDPT2) following the state-averaged CASSCF/(aug)-cc-pVDZ calculations is used to estimate the vertical S0-S1 excitation energies for all chromophore species. An accuracy of this approach is validated by comparing the computed estimates of the S0-S1 absorption maximum of the closely related chromophore from the DsRed protein to the known experimental value in the gas phase. An influence of the CASSCF active space on the aug-MCQDPT2 excitation energies is analyzed. The zwitterionic form of the asFP595 chromophore is found to be the most sensitive to a particular choice and amount of active orbitals. This observation is explained by the charge-transfer type of the S0-S1 transition involving the entire conjugated pi-electron system for the zwitterionic protonation state. According to the calculation results, the anionic form in the trans conformation is found to possess the most red-shifted absorption band with the maximum located at 543 nm. The bands of the zwitterionic and neutral forms are considerably blue-shifted compared to those of the anionic form. These conclusions are at variance with the results obtained in the TDDFT approximation for the asFP595 chromophore. The absorption wavelengths computed in the aug-MCQDPT2/CASSCF theory are as follows: 543 (535), 470 (476), and 415 (417) nm for the anionic, zwitterionic, and neutral forms of the trans and cis (in parentheses) isomers of the asFP595 chromophore. These data can be used as a reference for further theoretical studies of the asFP595 chromophore in different media and for modeling photoabsorption properties of the asFP595 fluorescent protein.

Chromophore Structure of the Kindling Fluorescent Protein asFP595 from Anemonia sulcata

Autocatalytic chromophore synthesis within the kindling fluorescent protein asFP595 is accompanied by a polypeptide chain break near the chromophore center. Tandem mass spectral analysis of the terminal proteolytic peptides adjacent to the asFP595 chain break allowed the elucidation of the exact structure of the asFP595 chromophore. The obtained results suggest that the mature asFP595 chromophore originates from a hydrolysis reaction of an intermediate acylimine-substituted structure.

Photoswitching of the Fluorescent Protein asFP595: Mechanism, Proton Pathways, and Absorption Spectra

Photoswitching of the Fluorescent Protein asFP595: Mechanism, Proton Pathways, and Absorption Spectra

Photoswitching of the Fluorescent Protein asFP595: Mechanism, Proton Pathways, and Absorption Spectra

Molecular light-switch: Off–on switching of the fluorescence of the protein asFP595 involves a trans–cis isomerization. Mixed quantum/classical simulations elucidate the spectroscopic properties of asFP595 and give detailed insights into the photoswitching mechanism. The conformational trans–cis switching triggers a proton-transfer cascade between the chromophore and adjacent amino acids. Supporting information for this article is available on the WWW under http://www.wiley-vch.de/contents/jc_2002/2007/z602315_s.pdf or from the author. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Ground-State Structures and Vertical Excitations for the Kindling Fluorescent Protein asFP595

Geometry configurations of a large fraction of the kindling fluorescent protein asFP595 around the chromophore region were optimized by using the effective fragment potential quantum mechanical-molecular mechanical (QM/MM) method. The initial coordinates of heavy atoms were taken from the structure from the Protein Data Bank archive corresponding to the dark-adapted state of the Ala143 --> Gly mutant of asFP595. Optimization of geometry parameters was performed for all internal coordinates in the QM part composed of the chromophore unit and the side chains of His197, Glu215, and Arg92 as well as for positions of effective fragments constituting the MMpart. The structures corresponding to the anion trans, anion cis, and zwitterion trans moieties were considered among various alternatives for the chromophore unit inside the protein matrix. The QM/MM simulations show that the protein environment provides stabilization for the trans-zwitterion isomer compared to the gas-phase conditions. By using the multiconfigurational CASSCF and the time-dependent density functional theory calculations, we estimated positions of spectral bands corresponding to vertical S(0)-S(1) transitions. The results of simulations support the assumption that the dark state of asFP595 corresponds to the anionic or zwitterionic trans-conformation, while the kindled state corresponds to the anionic cis-conformation.

Trans and cis Chromophore structures in the kindling fluorescent protein asFP595

The ab initio QM/MM calculations are used to optimize geometry configurations of the chromophore and surrounding residues for the kindling protein asFP595. The time-dependent DFT method is applied to estimate parameters of the S 0–S 1 vertical transition of the chromophore at the protein geometry taking into account effects from the nearest residues. The results of simulations provide a theoretical support to the hypothesis on the possibility of trans–cis izomerization of the chromophore in the mechanism of kindling. The system can absorb light in the trans anion form of the chromophore and emit at longer wavelength in the cis anion form.

Electronic Excitations of the Chromophore from the Fluorescent Protein asFP595 in Solutions.

We present the results of modeling spectral properties of the chromophore, 2-acetyl-4-(p-hydroxybenzylidene)-1-methyl-5-imidazolone (AHBMI), from the newly discovered fluorescent protein asFP595 in different solvents and compare computational and recent experimental data. The time-dependent density functional theory (TDDFT) method is used to estimate positions of spectral bands with large oscillator strengths for vertical transitions to excited states following geometry optimizations of chromophore coordinates in vacuo and in solutions. The performance of different TDDFT functionals in computing excitations for a simpler chromophore from the green fluorescent protein was tested at the preliminary stage. Properties of various protonation states (neutral, anionic, zwitterionic) for the cis and trans conformations of AHBMI are compared. By using the polarizable continuum model, the following solvents have been considered for AHBMI: water, ethanol, acetonitrile, and dimethyl sulfoxide. It is shown that the bands found experimentally in aqueous solution refer to the cis neutral and cis anionic (or trans zwitterionic) conformations. The computed band positions deviate from experimental ones in water by no more than 35 nm (0.23 eV). In accord with experimental studies, the band shifts in different solvents do not show correlation with the dielectric constant or dipole moment; however, the computed values of the shifts are much smaller than those measured experimentally for the ionic species.