Improving the design of the triple-decker motif in red fluorescent proteins

M. Khrenova, I. Polyakov, B. L. Grigorenko, A. I. Krylov, and A. V. Nemukhin
J. Phys. Chem. B. 121, 10602 – 10609 (2017)

We characterize computationally a fluorescent protein from red fluorescent proteins (RFP) containing 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, 1743−1747], 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 , 508–517] attempted to realize the triple-decker motif and developed an RFP variant called mRojoA-VYGV with two Tyr residues surrounding Chro. The crystal structure showed that only a Tyr-Chro pair was involved in pi-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 chromophore 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 the advanced 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.

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