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.