Triple-Decker Motif for Red-Shifted Fluorescent Protein Mutants

Among fluorescent proteins (FPs) used as genetically encoded fluorescent tags, the red-emitting FPs are of particular importance as suitable markers for deep tissue imaging. Using electronic structure calculations, we predict a new structural motif for achieving red-shifted absorption and emission in FPs from the GFP family. By introducing four point mutations, we arrive to the structure with the conventional anionic GFP chromophore sandwiched between two tyrosine residues. Contrary to the existing red FPs in which the red shift is due to extended conjugation of the chromophore, in the triple-decker motif, the chromophore is unmodified and the red shift is due to π-stacking interactions. The absorption/emission energies of the triple-decker FP are 2.25/2.16 eV, respectively, which amounts to shifts of ∼40 (absorption) and ∼25 nm (emission) relative to the parent species, the I form of wtGFP. Using a different structural motif based on a smaller chromophore may help to improve optical output of red FPs by reducing losses due to radiationless relaxation and photobleaching

Unusual Emitting States of the Kindling Fluorescent Protein: Appearance of the Cationic Chromophore in the GFP Family

The kindling fluorescent protein (KFP), the Ala143Gly variant of the natural chromoprotein asFP595, is a prospective biomarker in live cells. Following the results of QM/MM calculations, we predict that excitation of the protein under certain conditions, favoring formation of KFP fractions with the neutral chromophore, should result in fluorescence from the cationic form of the chromophore which is unusual for the members of the green fluorescent protein family. Occurrence of the neutral form is due to a water wire connecting the chromophore with the exterior of the protein. Occurrence of the cationic form is due to the excited-state proton transfer from the conserved Glu215 to the imidazolinone ring nitrogen of the chromophore. The emission band from conformations with the trans cationic chromophore should be noticeably shifted to the blue side around 520 nm compared to the well-known red fluorescence around 600 nm arising from the cis anionic species

A Light-Induced Reaction with Oxygen Leads to Chromophore Decomposition and Irreversible Photobleaching in GFP-Type Proteins

Photobleaching and photostability of proteins of the green fluorescent protein (GFP) family are crucially important for practical applications of these widely used biomarkers. On the basis of simulations, we propose a mechanism for irreversible bleaching in GFP-type proteins under intense light illumination. The key feature of the mechanism is a photoinduced reaction of the chromophore with molecular oxygen (O₂) inside the protein barrel leading to the chromophore’s decomposition. Using quantum mechanics/molecular mechanics (QM/MM) modeling we show that a model system comprising the protein-bound Chro^– and O₂ can be excited to an electronic state of the intermolecular charge-transfer (CT) character (Chro^•···O₂^–•). Once in the CT state, the system undergoes a series of chemical reactions with low activation barriers resulting in the cleavage of the bridging bond between the phenolic and imidazolinone rings and disintegration of the chromophore

Improving the Design of the Triple-Decker Motif in Red Fluorescent Proteins

We characterize computationally a red fluorescent protein (RFP) with the chromophore (Chro) sandwiched between two aromatic tyrosine rings in a triple-decker motif. According to the original proposal [J. Phys. Chem. Lett. 2013, 4, 1743], such a tyrosine-chromophore-tyrosine π-stacked construct can be accommodated in the green fluorescent protein (GFP). A recent study [ACS Chem. Biol. 2016, 11, 508] attempted to realize the triple-decker motif and obtained an RFP variant called mRojoA-VYGV with two tyrosine residues surrounding the chromophore. The crystal structure showed that only a tyrosine-chromophore pair was involved in π-stacking, whereas the second tyrosine was oriented perpendicularly, edge-to-face with respect to the chromophore. We propose a more promising variant of this RFP with a perfect triple-decker unit achieved by introducing additional mutations in mRojoA-VYGV. The structures and optical properties of model proteins based on the structures of mCherry and mRojoA are characterized computationally by QM­(DFT)/MM. The electronic transitions in the protein-bound chromophores are computed by high-level quantum chemical methods. According to our calculations, the triple-decker chromophore unit in the new RFP variant is stable within the protein and its optical bands are red-shifted with respect to the parent mCherry and mRojoA species

First-Principles Characterization of the Energy Landscape and Optical Spectra of Green Fluorescent Protein along the A→I→B Proton Transfer Route

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

Toward Molecular-Level Characterization of Photoinduced Decarboxylation of the Green Fluorescent Protein: Accessibility of the Charge-Transfer States

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. <i>Nature Struct. Biol.</i> <b>2002</b>, <i>9</i>, 37–41; Bell et al. <i>J. Am. Chem. Soc.</i> <b>2003</b>, <i>125</i>, 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