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

Abstract

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.

Dipole Moment Variation Clears Up Electronic Excitations in the π-Stacked Complexes of Fluorescent Protein Chromophores

“…17,18,23 Besides experimental efforts, molecular simulations also contributed to this field. 24,25 The π-stacking-induced spectral shifts in chromophores of the fluorescent proteins usually do not exceed 0.2 eV. This amount is comparable with an expected accuracy of modern quantum chemical methods in computing transition energies between the lowest electronic states.…”

“…Two quantum chemistry methods were utilized to obtain the electron density in the ground and excited states. First, the CASSCF approach is a method of choice when considering electronic excitations in organic chromophores. ,, Following our previous experience with the chromophores under study, , we rely on the distribution of 12 electrons over 12 active molecular orbitals and the density averaging over two lowest electronic states, S 0 and S 1 (sa2-CASSCF­(12/12)). The second option is the TDDFT approach; in this work, we employ the CAM-B3LYP, ωB97X-D3, and PBE0 functionals.…”

“…The first successful attempt in this field was a replacement Thr203Tyr in GFP that resulted in a new yellow color of fluorescence and a bathochromic shift of the absorption band . Later, the same idea was applied to fluorescent proteins with the elongated chromophores from the DsRed-like proteins. ,, Besides experimental efforts, molecular simulations also contributed to this field. , …”

Computational Challenges in Modeling of Representative Bioimaging Proteins: GFP-Like Proteins, Flavoproteins, and Phytochromes

“…The computational protocol PBE-D3/TZV2P-MOLOPT-GTH was used in QM, and the AMBER force field, in MM. The simulations showed that different starting conformations converged to similar structures with the triple-decker motif (see Figure 6 in ref ).…”

“…On the basis of careful analysis of the protein structure of mRojoA, we suggested a series of mutations capable of fixing Tyr63 in a coplanar orientation with respect to the chromophore, along with the previously achieved π-stacking of the chromophore with Tyr197 (Figure ). Each replacement had a specific purpose.…”