Multiple phenylalanine derivatives containing substitutions at the ortho and meta positions were incorporated at the Y66 position in the chromophore of superfolder green fluorescent protein (sfGFP) using a broadly permissive pyrrolysyl-tRNA synthetase mutant in conjunction with an orthogonal amber suppressor tRNA. Phenylalanine derivatives containing aldehyde, bromo-, chloro-, trifluoromethyl-, ketone, methyl-, methoxy-, and nitrile functional groups at the ortho or meta positions were substituted into the sfGFP chomophore in the current study. The mutated chromophores displayed absorbance maxima ranging from 311 to 423 nm and fluorescence maxima ranging from 386 to 476 nm for these mutant proteins, which is a significant shift in absorbance and emission maxima compared to the wildtype protein (λexc 485 nm λem 510 nm). Two of the phenylalanine derivatives resulted in chromophores that emitted in the visible region of the electromagnetic spectrum. The meta-methoxy (λexc 374 nm and λem 450 nm) chromophore emitted a blue light that could be seen with the naked eye when irradiated with UV light. The ortho-methoxy (λexc 423 nm and λem 476 nm) chromophore emitted a yellow light that could be seen by the naked eye under white light. The larger Stokes shift of these mutant proteins compared to wildtype sfGFP as well as the shift in absorbance and emission maxima will provide new tools for FRET and fluorescence imaging studies.

Spectroscopic properties of the GFP chromophore containing substituted phenylalanine derivatives in place of tyrosine

249th National Meeting (2015)

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