We present the results of quantum chemical calculations of the electronic properties of the anionic form of the green fluorescent protein chromophore in the gas phase. The vertical detachment energy of the chromophore is found to be 2.4-2.5 eV, which is below the strongly absorbing ππ* state at 2.6 eV. The vertical excitation of the lowest triplet state is around 1.9 eV, which is below the photodetachment continuum. Thus, the lowest bright singlet state is a resonance state embedded in the photodetachment continuum, whereas the lowest triplet state is a regular bound state. Based on our estimation of the vertical detachment energy, we attribute a minor feature in the action spectrum as due to the photodetachment transition. The benchmark results for the bright ππ* state demonstrated that the scaled opposite-spin method yields vertical excitation within 0.1 eV (20 nm) from the experimental maximum at 2.59 eV (479 nm). We also report estimations of the vertical excitation energy obtained with the equation-of-motion coupled cluster with the singles and doubles method, a multireference perturbation theory corrected approach MRMP2 as well as the time-dependent density functional theory with range-separated functionals. Expanding the basis set with diffuse functions lowers the ππ* vertical excitation energy by 0.1 eV at the same time revealing a continuum of "ionized" states, which embeds the bright ππ* transition.

We present the results of quantum chemical calculations of the transition energies and conical intersection points for the two lowest singlet electronic states of the green fluorescent protein chromophore, 4'-hydroxybenzylidene-2,3-dimethylimidazolinone, in the vicinity of its cis conformation in the gas phase. Four protonation states of the chromophore, i.e., anionic, neutral, cationic, and zwitterionic, were considered. Energy differences were computed by the perturbatively corrected complete active space self-consistent field (CASSCF)-based approaches at the corresponding potential energy minima optimized by density functional theory and CASSCF (for the ground and excited states, respectively). We also report the EOM-CCSD and SOS-CIS(D) results for the excitation energies. The minimum energy S0/S1 conical intersection points were located using analytic state-specific CASSCF gradients. The results reproduce essential features of previous ab initio calculations of the anionic form of the chromophore and provide an extension for the neutral, cationic, and zwitterionic forms, which are important in the protein environment. The S1 PES of the anion is fairly flat, and the barrier separating the planar bright conformation from the dark twisted one as well as the conical intersection point with the S0 surface is very small (less than 2 kcal/mol). On the cationic surface, the barrier is considerably higher (∼13 kcal/mol). The PES of the S1 state of the zwitterionic form does not have a planar minimum in the Franck-Condon region. The S1 surface of the neutral form possesses a bright planar minimum; the energy barrier of about 9 kcal/mol separates it from the dark twisted conformation as well as from the conical intersection point leading to the cis-trans chromophore isomerization.

Structures and optical spectra of the green fluorescent protein (GFP) forms along the proton transfer route A→I→B are characterized by first-principles calculations. We show that in the ground electronic state the structure representing the wild-type (wt) GFP with the neutral chromophore (A-form) is lowest in energy, whereas the systems with the anionic chromophore (B- and I-forms) are about 1 kcal/mol higher. In the S65T mutant, the structures with the anionic chromophore are significantly lower in energy than the systems with the neutral chromophore. The role of the nearby amino acid residues in the chromophore-containing pocket is re-examined. Calculations reveal that the structural differences between the I- and B-forms (the former has a slightly red-shifted absorption relative to the latter) are based not on the Thr203 orientation, but on the Glu222 position. In the case of wt-GFP, the hydrogen bond between the chromophore and the His148 residue stabilizes the structures with the deprotonated phenolic ring in the I- and B-forms. In the S65T mutant, concerted contributions from the His148 and Thr203 residues are responsible for a considerable energy gap between the lowest energy structure of the B type with the anionic chromophore from other structures.

Electronic structure calculations of the singly and doubly ionized states of deprotonated 4(')-hydroxybenzylidene-2,3-dimethylimidazolinone (HBDI anion) are presented. One-electron oxidation produces a doublet radical that has blueshifted absorption, whereas the detachment of two electrons yields a closed-shell cation with strongly redshifted (by about 0.6 eV) absorption relative to the HBDI anion. The results suggest that the doubly oxidized species may be responsible for oxidative redding of green fluorescent protein. The proposed mechanism involves two-step oxidation via electronically excited states and is consistent with the available experimental information [A. M. Bogdanov, A. S. Mishin, I. V. Yampolsky, et al., Nat. Chem. Biol. 5, 459 (2009)]. The spectroscopic signatures of the ionization-induced structural changes in the chromophore are also discussed.

We present quantum chemical calculations of the properties of the anionic form of the green fluorescent protein (GFP) chromophore that can be directly compared to the results of experimental measurements: the cis-trans isomerization energy profile in water. Calculations of the cis-trans chromophore isomerization pathway in the gas phase and in water reveal a problematic behavior of density functional theory and scaled opposite-spin-MP2 due to the multiconfigurational character of the wave function at twisted geometries. The solvent effects treated with the continuum solvation models, as well as with the water cluster model, are found to be important and can reduce the activation energy by more than 10 kcal/mol. Strong solvent effects are explained by the change in charge localization patterns along the isomerization coordinate. At the equilibrium, the negative charge is almost equally delocalized between the phenyl and imidazolin rings due to the interaction of two resonance structures, whereas at the transition state the charge is localized on the imidazolin moiety. Our best estimate of the barrier obtained in cluster calculations employing the effective fragment potential-based quantum mechanics/molecular mechanics method with the complete active space self-consistent field description of the chromophore augmented by perturbation theory correction and the TIP3P water model is 14.8 kcal/mol, which is in excellent agreement with the experimental value of 15.4 kcal/mol. This result helps to resolve previously reported disagreements between experimental measurements and theoretical estimates.

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Irradiation of the green fluorescent protein (GFP) by intense violet or UV light leads to decarboxylation of the Glu222 side chain in the vicinity of the chromophore (Chro). This phenomenon is utilized in optical highlighters, such as photoactivatable GFP (PA-GFP). Using state-of-the-art quantum chemical calculations, we investigate the feasibility of the mechanism proposed in the experimental studies [van Thor et al. Nature Struct. Biol.2002, 9, 37-41; Bell et al. J. Am. Chem. Soc.2003, 125, 37-41]. It was hypothesized that a primary event of this photoconversion involves population of a charge-transfer (CT) state via either the first excited state S1 when using longer wavelength (404 and 476 nm) or a higher excited state when using higher energy radiation (254 and 280 nm). Based on the results of electronic structure calculations, we identify these critical CT states (produced by electron transfer from Glu to electronically excited Chro) and show that they are accessible via different routes, i.e., either directly, by one-photon absorption, or through a two-step excitation via S1. The calculations are performed for model systems representing the chromophore and the key nearby residues using two complementary approaches: (i) the multiconfigurational quasidegenerate perturbation theory of second order with the occupation restricted multiple active space scheme for configuration selection in the multiconfigurational self-consistent field reference; and (ii) the single-reference configuration interaction singles method with perturbative doubles that does not involve active space selection. We examined electronic transitions with nonzero oscillator strengths in the UV and visible range between the electronic states involving the Chro and Glu residues. Both methods predict the existence of CT states with nonzero oscillator strength in the UV range and a local excited state of the chromophore accessible via S1 that may lead to the target CT state. The results suggest several possible scenarios for the primary photoconversion event. We also demonstrate that the point mutation Thr203His exploited in PA-GFP results in shifting the light wavelength to access the CT up to 20 nm, which suggests a possibility of a rational design of photoactivatable proteins in silico.

We present the results of high-level electronic structure and dynamics simulationsof the photoactive protein Dreiklang. With the goal to understand the details ofDreiklang's photocycle, we carefully characterize the excited states of the ON-andOFF-forms of Dreiklang. The key ?nding of our study is the existence of a lowlyingexcited state of a charge-transfer (CT) character in the neutral ON form andthat population of this state, which is nearly isoenergetic with the locally excited(LE) bright state, initiates series of steps ultimately leading to the formation ofthe hydrated dark chromophore (OFF state). These results allow us to re?ne themechanistic picture of the Dreiklang's photocycle and photoactivation. File list (3) download file view on ChemRxiv dreiklang_paper_draft.pdf (2.19 MiB) download file view on ChemRxiv dreiklang_si_draft.pdf (3.21 MiB) download file view on ChemRxiv Dreiklang-hydration.zip (15.42 MiB)